KR19990064155A - A larval hormone or an agonist thereof as a chemical ligand for regulating gene expression in plants by receptor mediated transactivation - Google Patents

Abstract

The present invention relates to a method for regulating gene expression in a plant. Specifically, the methods of the invention include transforming plants using USP receptor expression cassettes encoding USP receptors and one or more target expression cassettes encoding the target polypeptides. The expression of the target polypeptide is activated in the presence of the USP receptor polypeptide by contacting the transformed plant with a lectin hormone or an agonist thereof. Optionally, additional " second " receptor expression cassettes encoding the USP and other receptor polypeptides may be used. The method of the present invention is useful for controlling various agricultural important properties such as plant fertilization. Also described is a method for identifying a ligand for a USP that is not known in advance. The test substance is identified by contacting the test substance with a plant cell transformed with a USP receptor expression cassette and a target expression cassette. The target expression cassette encodes a reporter polypeptide whose expression can be quantitatively or qualitatively measured whereby the test substance is identified as a ligand for USP.

Description

A larval hormone or an agonist thereof as a chemical ligand for regulating gene expression in plants by receptor mediated transactivation

The present invention relates to the chemical regulation of gene expression in plants. In particular, the present invention relates to methods of regulating receptor-mediated expression of a target polypeptide in a plant cell using a larval hormone or agonist thereof as a chemical ligand, as well as a method of regulating transgenic plant cells containing a suitable expression cassette, Or plants and their offspring.

In some cases, it is desirable to regulate the expression time or degree of expression of phenotypic traits in plants, plant cells or plant tissues. The ideal situation is to control the manifestation of such properties caused by chemicals that can be easily applied to packaged crops, shrubs, and the like. One such system for regulating gene expression that can be used to achieve ideal conditions is the steroid and thyroid hormone phase (superfamily) of nuclear receptors, as it is not yet known to be naturally present in plants. Steroids and thyroid hormone phases of nuclear receptors are found in mammals and insects, and consist of more than 100 known proteins. Some of the receptors found in mammals are the retinoic acid receptor (RAR), the vitamin D receptor (VDR), the thyroid hormone receptor (T ₃ R) and the retinoic acid X receptor (RXR). The receptors and other receptors in the phase bind to the 5 ' regulatory region of the target gene and transactivate the target gene expression as the chemical ligand binds to the receptor.

In addition to the receptors found in the mammals described above, receptors with similar structures and activities have been identified in the insect Drosophila (Koelle et al., Cell 67: 599 (1991); Christianson and Kafatos, Biochem. Biophys. Res. Comm. 193: 1318 (1993); Henrich et al., Nucleic Acids Res. 18: 4143 (1990)). The Ecdysone receptor (EcR) binds the insect steroid hormone 20-hydroxyexidone and transactivates gene expression when heterodimerized with the product of the Ultraspiracle gene (USP). In addition, other chemical ligands, such as hormone agonists other than 20-hydroxyexciton, will bind to EcR under similar conditions and induce transactivation of the target gene.

In addition, USP has been found to be a member of the nuclear receptor's steroid and thyroidal phase, even though its ligand has not been identified and thus is considered to be an "orphan" receptor (Seagraves, Cell 67: 225-228 (1991) ]. USP relates to the sequence RXRa (Oro et al., Nature, 347: 298-301 (1990)), and RXRs can form heterodimers with EcR (Thomas et al., Nature 362: 471-475 (1993)]. The larvae hormone agonist methoprene and its derivatives methoprenic acid have been shown to activate transcriptionally by recombinant reporter genes in both insect and mammalian cells by activation by RXRa (Harmon et al., Proc. Natl. Acad. Sci. 92: 6157-6160 (1995)). However, the larval hormone does not induce RXRα-mediated transactivation (Harmon et al.). The orbital receptor USP has been suggested as potent (Harmon et al .: Seagraves; Oro et al., Current Opinion in Genetics and Development 2: 269-274 (1992)], to date there is no clear evidence for a nuclear larval hormone receptor (Harmon et al .; Henrich and Brown, Insect Biochem. Molec. Biol. 25: 881-897 (1995)).

Because these chemicals are already known for use in agriculture, larval hormones and their agonists provide a previously unrecognized opportunity for chemical regulation of gene expression in plants. What is lacking so far is the means by which these chemicals can be used to induce the transactivation of target genes in transgenic plants. In accordance with the present invention, it has been found that the larval hormone and its derivatives are ligands to the orbital receptor USP. This finding has led to genetic control strategies for plants using nuclear receptors that are not naturally present in plants. This means that only the application effect of the larval hormone or its agonist can induce the expression of the genetically engineered target gene. As demonstrated in the present invention, a USP receptor polypeptide and a plant-expressible gene encoding it regulate expression of the target polypeptide by acting on the plant cell, wherein the USP receptor polypeptide binds to the target expression < RTI ID = 0.0 > Activate the 5 'control region of the cassette. This method of regulating gene expression in plants is useful for controlling several agronomically important traits such as plant modifications.

The present invention relates to a method of regulating gene expression in a plant. Specifically, the methods of the present invention are directed to a USP receptor expression cassette encoding a USP receptor polypeptide, one or more target expression cassettes encoding the target polypeptide, and optionally a second receptor expression cassette encoding a second receptor polypeptide different from the USP receptor polypeptide And planting the plant. The expression of the target polypeptide is activated or inhibited in the presence of the USP receptor polypeptide by contacting the transformed plant with the larval hormone or agonist thereof. Optionally, an additional " second " receptor expression cassette can be used, wherein the second receptor expression cassette encodes a different receptor polypeptide than USP. The method of the present invention is useful for controlling various agricultural important properties such as plant fertilization.

The present invention also relates to a transgenic plant comprising a USP receptor expression cassette and a target expression cassette which can be activated by a larval hormone or agonist thereof. The present invention also encompasses ligands for USP that are not known to be effective under the plant cell environment. The test substance is identified by contacting the plant cell transformed with the USP receptor expression cassette and the target expression cassette with the test substance. The target expression cassette encodes a receptor polypeptide whose expression can be quantitatively or qualitatively determined whereby the test substance is identified as a ligand for USP.

Figure 1 provides a schematic representation of a plant cell comprising a VP16-USP receptor expression cassette and a target expression cassette with a direct repeat response element present in the 5 ' regulatory region. In the presence of the larval hormone or agonist thereof, the VP16-USP receptor activates the expression of the target polypeptide.

Figure 2 provides a schematic of plant cells comprising both the USP-VP16 and GAL4-EcR receptor expression cassettes. Upon exposure to the larval hormone or agonist thereof, the activation of the expression of the target polypeptide caused by the combination of the GAL4-EcR and USP-VP16 receptor polypeptides is reversed.

Figure 3 corresponds to Figure 2, except that the chemical ligand tebufenozide (also known as RH 5992) is present. In the presence of the larval hormone or agonist thereof, activation is also reversed under these circumstances.

"Larvae hormone" refers to a class of chemical compounds produced by insects. The larval hormone regulates larval metamorphosis by inducing maintenance of insect larval characteristics and subsequent maturation prevention. The action of larval hormones is biologically manifested as behavioral, biochemical or molecular effects. Some naturally occurring larval hormones are isolated and characterized.

The agonist agonist means a class of compounds that exhibits one or more of the biological activities of the larval hormone. The agonist agonist may not be structurally similar or similar to the larval hormone. Such techniques also include compounds that may be metabolic precursors of compounds that directly produce the above-mentioned larval hormone-like biological effects. For example, methoprene is the metabolic precursor of methoprenic acid, which in turn directly indicates the larval hormone-like biological effects observed by applying methoprene.

The term " receptor polypeptide " as used herein refers to a polypeptide capable of activating or inhibiting the expression of a target polypeptide in response to an applied chemical ligand. The receptor polypeptide consists of a ligand binding domain, a DNA binding domain and a transactivation domain. The ligand binding domain comprises an amino acid sequence having a structure that noncovalently binds a complementary chemical ligand. The DNA binding domain comprises an amino acid sequence that non-covalently binds a specific nucleotide sequence known as a response element (RE). The at least one reactive element is located within the 5 ' regulatory region of the target expression cassette. Each RE comprises a pair of half-sites, each half-site having 5 to 6 base pairs of cores in which a single DNA binding domain recognizes a single half-site. The half-sites can be arranged in a relatively linear orientation with respect to each other as direct repeats, crossings repeats or inverted repeats. The nucleotide sequence, space and linear orientation of the half-site determines whether the DNA binding domain or domains will form a complementary binding pair with the response element. The transactivation domain comprises one or more sequences of amino acids that act as an < RTI ID = 0.0 > subdomain < / RTI > that affects the effect of the transcription factor upon preincubation and combination in the TATA box. The effect of the transactivation domain allows for repeated transcription initiation frequencies leading to greater levels of gene expression.

A " receptor expression cassette " comprises a nucleotide sequence for a 5 'regulatory region operably linked to a nucleotide sequence encoding a receptor polypeptide and a 3' terminator region (a stop codon and a polyadenylation sequence) compared to the receptor. The 5 ' regulatory region can promote expression in plants.

&Quot; USP " refers to the receptor ultra spiracle found in Drosophila. This is also known as " XR2C ", isolated and cloned, and its ligand-binding domain has been identified by sequence homology with known ligand binding domains (Henrich et al., Nucleic Acids Research 18: 4143-4148 1990), the chemical ligands or ligands that bind thereto are not known to date. As used herein, the term " USP " means a naturally occurring form of the receptor, and mutant or chimeric forms thereof. This includes, but is not limited to, the chimeric forms of the USP, which include at least the mutant or chimeric forms described herein, and the ligand binding domains of native USP and mutants thereof. In the present invention, more than one type of USP can be used at the same time.

A " second receptor expression cassette " comprises a nucleotide sequence for a 5 'regulatory region operably linked to a nucleotide sequence encoding a USP that is operably linked to a 3' terminus region and a different receptor polypeptide. Second receptor expression cassettes include, but are not limited to, EcR, RXR, DHR38 (Kafatos et al., Proc. Natl. Acad. Sci. 92: 7966-7970 (1995)] and mutants and chimeric forms thereof.

&Quot; Residue " means the portion of the receptor polypeptide derived from the indicated source. For example, " USP-residue " refers to a portion of a receptor polypeptide derived from a natural ultraspiracle receptor. The residues used herein may comprise one or more domains and the residues at least comprise the ligand binding domain of the named receptor.

The term " chimera " is used to indicate that the receptor is composed of one or more domains with heterogeneous origin for the other domains present. By " heterologous " is meant that one or more domains present in the receptor polypeptide differ in their natural origin relative to the other domains. For example, if the transactivation domain from the herpes simplex VP16 protein is operably linked to a USP receptor from the drosophila, the VP16 transactivation domain is heterologous to the USP-residue. Additionally, if a domain from the USP is operatively linked to a domain from the RXR to provide a functional receptor, the chimeric fusion will have a domain that is heterologous to each other. These chimeric receptor polypeptides are encoded by operably linked nucleotide sequences to generate coding sequences that are not naturally present. Chimeric receptor polypeptides of the invention are referenced by linear nomenclature from the N-terminal to the C-terminal portion of the polypeptide. Using this nomenclature, a chimeric receptor polypeptide having a transactivation domain from VP16 added to the N-terminal region of the USP receptor will be named VP16-USP. Conversely, if VP16 is added to the C-terminal region of the USP receptor, the chimeric receptor polypeptide will be designated USP-VP16.

The gene construct is named by the 5 ' regulatory region and its operably-linked coding sequence, with the 5 ' regulatory region preceding the slash mark (/) and the coding sequence after the slash mark. For example, the gene construct 35S / USP-VP16 represents the 35S promoter of Cauliflower Mosaic Virus operably linked to the DNA encoding the chimeric receptor USP-VP16, C-terminal region. If a receptor polypeptide is referred to, no promoter is named. For example, the gene construct encodes a USP-VP16 polypeptide.

A "target expression cassette" comprises a nucleotide sequence for a 5 'regulatory region operably linked to a nucleotide sequence encoding a target polypeptide, wherein the expression of the target polypeptide is activated or inhibited by the receptor polypeptide in the presence of a chemical ligand. The 5 'regulatory region of the target gene comprises the core promoter sequence, the transcription initiation sequence, and the response or response elements required for complementary binding of the receptor polypeptide. The 5 ' regulatory region can promote expression in plants. Also, the target expression cassette comprises a 3 'termination region (stop codon and polyadenylation sequence).

The larval hormones I, II, III and O are known to be a substituted variant of I (Fundamentals of Insect Physiology, M. S. Blum, Ed., John Wiley & Sons, New York, 1985). The larval hormone I is methyl 10,11-epoxy-7-ethyl-3,11-dimethyl-trans-2,6-tridecadienoate. The structure of larval hormone I is as follows.

The larval hormone antagonist is a compound that exhibits one or more of the biological activities of the larval hormone. Compounds having such properties that are structurally related to larval hormones include, but are not limited to, quinoprene, methoprene, hydroprene, and methoprenic acid. The structure of quinoprene as an example of these larval hormone agonists is as follows.

In addition, a larval hormone agonist that is not structurally related to the larval hormone is known. Such compounds include, but are not limited to, the known larval hormone agonists polycyclic, non-isoprenoid compounds phenoxycarb. The formula for phenoxycarb is ethyl [2- (4-phenoxyphenoxy) ethyl] carbamate. The structural formula of phenoxycarb is shown below.

Another hyperglycemic agonist useful in the present invention is the compound diophenolane, diphenyl ether compound. The formula for diophenolane is 4- (2-ethyl-1,3-dioxolan-4-ylmethoxy) phenyl phenyl ether. This compound is known to have larval hormone activity and to function similarly to the function of the compound phenoxycarb or methoprene.

The use of larval hormone agonists in the present invention provides several advantages. First, these compounds can be readily synthesized and readily available. Second, the majority of these compounds have the advantage of being already tested for agricultural productivity, and such compounds are easy to use for packaging applications in crops.

The present invention utilizes the discovery that the larval hormone or agonist acts as a chemical ligand for the USP receptor. The present invention has developed a novel method for regulating gene expression in plants using chemical ligands for USP that have not been previously known in combination with plant-expressible USP receptor expression cassettes and appropriate target expression cassettes.

The majority of insect growth regulators have been shown to inhibit molting in insects and seem to work directly on receptors associated with initiation of molting. Such insect growth regulators include, but are not limited to, triflumuron [(1- (2-chlorobenzoyl) -3- (4-trifluoromethoxyphenyl) urea], hexamphlorum [1- [ (1,1,2,2-tetrafluoroethoxy) phenyl] -3- (2,6-difluorobenzoyl) urea], teflubenzuron [1- (Dichloro-2,4-difluorophenyl) -3- (2,6-difluorobenzoyl) urea], flufenoxuron [1- Fluoro-p-tolyloxy) -2-fluorophenyl] -3- (2,6-difluorobenzoyl) urea), flucyclosuron [1- [ (1,1,2,3,3-pentafluoro-benzylideneamino-oxy) -p-tolyl] -3- (2,6-difluorobenzoyl) urea] 3,3-hexafluoropropoxy) phenyl] -3- (2,6-difluorobenzoyl) urea]. Also included is an addition, including, but not limited to, diplubenolone and chlorflurozoline A benzoyl phenyl urea insecticides may be used in the present invention.

In addition, retinoic acid or derivatives thereof may be useful ligands for regulating gene expression in transgenic plants. Retinoic acid has recently been shown to activate heterologous-expressed USP gene products in cultured mammalian cell lines (Harmon et al.). Thus, an insect receptor can be regulated by applying a retinoic acid derivative to a transgenic plant comprising the appropriate combination of receptor (s) and target gene, as discussed below.

In addition, natural sources can be used as ligands to insect receptors expressed in transgenic plants such as transgenic maize and wheat. Many plants have been found to synthesize compounds that promote or inhibit insect secretion. For example, one of the most active ecdysteroids, myristone, is isolated from plant sources. Many plant classes are known to produce adic steroid or larval hormone activity.

In addition, gene expression can be regulated in transgenic plants using larval hormone agonists as ligands. Examples of such ligands are ethyl 4- [2, 3-butylcarboxyloxy] -benzoate; Methoxy-6- [1- (4-methoxyphenyl) ethyl] -1,3-benzodioxole or ethyl E-3-methyl-2-dodecenoate. Such ligands can be used to activate the expression of a gene having a low basal expression in a heterogeneous plant system expressing the modified insect receptor (s), or to suppress gene expression having a high basal expression.

The methods of the invention include transforming a plant cell or plant with a USP receptor expression cassette and a target expression cassette. Upon expression, the USP receptor polypeptide in the plant cell, plant, or progeny thereof obtained in the presence of the larval hormone or agonist thereof activates the 5 ' regulatory region of the target expressed cassette in the transgenic cell or plant (Fig. 1). The larval hormone or agonist thereof recognized herein as binding to the ligand binding domain of USP is essential to the present invention.

In addition, expression control of the target expression cassette can be achieved by randomly expressing USP and other additional second receptor polypeptides or polypeptides in plants (FIGS. 2 and 3). Examples of additional second receptor polypeptides included in the present invention include, but are not limited to, EcR, RXR, DHR38 (Kafatos et al., Proc. Natl. Acad. Sci. 92: 7966-7970 (1995)] and mutants or chimeric forms thereof. The use of these receptors to regulate ligand-induced transactivation is described in International Patent Application No. PCT / EP 96/00686, filed February 19, 1996, which is incorporated herein by reference.

The ligand-binding domain of a USP receptor polypeptide provides a means of chemically modulating the activation of the 5 ' regulatory region of the target expression cassette by the nematode hormone or agonist thereof. USP is similar to the steroid receptor RXRa with 9-cis-retinoic acid as a chemical ligand. In addition, the USP forms a heterodimer with an EcR receptor polypeptide, binds to EcR and expresses the expression of the target polypeptide in mouse kidney cells transformed in response to the application of the insect hormone, an insect hormone independent of the agonist hormone and its agonist ≪ / RTI > [WO 94/01558]. Receptor USP and its ligand-binding domain have been found in the present invention to be particularly useful for modulating expression of a target polypeptide in a plant by reacting with a larval hormone or an agonist thereof, as described in the Examples below.

A chimeric form of a USP receptor polypeptide can also be used in the present invention to activate the expression of a target polypeptide in the presence of a lormonal hormone or agonist thereof. The DNA binding or transactivation domains of chimeric USP receptor polypeptides can be selected from heterogeneous sources based on their efficiency for transactivation or DNA binding. Such domains of chimeric receptor polypeptides can be obtained from all organisms whose transcriptional control function is similar, for example, from plants, insects and mammals. In one aspect of the invention, these domains are selected from steroids of nuclear receptors and other members of the thyroid hormone phase. The use of chimeric receptor polypeptides has the advantage of combining domains from different sources. The chimeric USP receptor polypeptides provided herein have the advantage of combining the recognition of specific response elements with optimal transactivation activity or modified RE binding or ligand hormone or agonist thereof. Thus, a chimeric polypeptide produced for a particular purpose can be constructed. In addition, these chimeric receptor polypeptides provide improved functionality under the heterologous environment of plant cells.

It is also contemplated that the ligand-binding and DNA-binding domains can be assembled into chimeric receptor polypeptides in all functional arrangements as part of the present invention. For example, if one subdomain of the transactivation domain is found in the N-terminal portion of the naturally occurring receptor, the chimeric receptor polypeptide of the present invention may be used in place of the N-terminal subdomain or in addition to the N- And a transactivating domain at the C-terminus. In addition, the chimeric receptor polypeptides described herein may have multiple domains in the same form, for example, one or more transactivation domains (or two domains) per receptor polypeptide.

Thus, one aspect of the present invention provides USP receptor polypeptides that possess superior properties for activating and transactivating the expression of a target polypeptide in the presence of a lormonal hormone or agonist thereof. The transactivation domain can be defined as an amino acid sequence that increases the productive transcription initiation by RNA polymerase (see generally Ptashne, Nature 335: 683-689 (1988)). Different transactivation domains are known to have different degrees of potency in their ability to increase transcription initiation. In the present invention, in order to obtain a high level of target polypeptide expression in response to the presence of the larval hormone or its agonist, it is preferable to use a transactivation domain having excellent transactivation efficiency in plant cells. Transactivation domains that have been found to be particularly effective in the methods of the invention include, but are not limited to, VP16 (isolated from the herpes simplex virus). In one preferred embodiment of the invention, the transactivation domain from VP16 is operably linked to a USP-residue to produce a chimeric USP receptor polypeptide that modulates expression of the target polypeptide in the plant. In addition, other transactivating domains may be effective.

The DNA binding domain is a sequence of amino acids with specific functional properties due to the USP receptor polypeptide for a specific sequence of nucleotides (referred to as the response element) present in the 5 ' regulatory region of the target expression cassette. The structure of the DNA binding domain to the steroid and thyroid phase of the nuclear receptor is highly conserved from species to species and as a result limits mutation in the response elements used to form complementary binding pairs (Evans, Science 240: 889-895 (1988). Nevertheless, it is possible to introduce considerable flexibility in the way in which gene expression is regulated by using reactive elements in other ways. In a preferred embodiment of the invention, multiple copies and preferably 1 to 11 copies of a suitable response element are present in the 5 ' regulatory region, which binds multiple sites to the USP or any second receptor polypeptide, To indicate activation.

Additional flexibility in gene regulation methods can be achieved by changing the linear orientation or position of the response elements in the 5 ' control region. The response element recognized by the class II receptor protein has a " two minute " chromosome consisting of two "half-sites" (Evans, Science 240: 889-895 (1988)). Each receptor polypeptide binds to a " half-site ". These " half-sites " can be oriented in direct repetition, reversed repetition or phonetic form. In one aspect of the invention, one or more of the USP receptor polypeptide molecules recognize a direct repetitive (DR) response element, whereby activation of the target expression cassette is achieved in the presence of a lectin hormone or agonist thereof.

Additional flexibility in controlling gene expression by the present invention can be achieved by using DNA binding domains and reaction elements from other transcription activators, including, but not limited to, LexA or GAL4 proteins. this. The DNA binding domain from the LexA protein encoded by the lexA gene from E. coli and its complementary binding site (Brent and Ptashne, Cell 43: 729-736, (1985), LexA / GAL4 transcriptional activator Quot;). ≪ / RTI > Another useful source is from yeast GAL4 protein (Sadowski et al. Nature 335: 563-564 (1988), described as GAL4-VP16 transcription activator]. In a preferred embodiment of the invention, the chimeric modification of any second receptor polypeptide is constructed by fusing the GAL4 DNA binding domain to a residue containing a ligand binding domain from an EcR.

In addition, the 5 ' regulatory regions of the USP and any second receptor expression cassette contain promoters and are expressed in plant tissues and cells. Suitable promoters are selected for the receptor expression cassette so that the expression of the receptor polypeptide can be constitutively and orally regulated and can be tissue-specific, cell-specific or cellular complement-specific. In addition, the expression of the receptor polypeptide itself can be chemically induced in plants by selecting a promoter, thereby increasing the level of promoter induction by the ligand. By combining the promoter elements that provide chemically induced expression and provide for specific expression, the receptor polypeptide can be expressed or activated in plant specific cells or tissues that respond to the chemical application.

Nucleotide sequences encoding receptor polypeptides can be modified for improved expression, improved functionality, or both in plants. Such modifications include, but are not limited to, use of modified codons, insertion of introns, or construction of mutants. In one aspect of the invention, an expression cassette comprising a drug-specific or pistil-specific promoter operably linked to a nucleotide sequence encoding a USP receptor polypeptide is used to express the target polypeptide < RTI ID = 0.0 > ≪ / RTI >

Also described are target polypeptides whose expression is activated by a receptor polypeptide in the presence of a nematicide or agonist thereof. The expression of all coding sequences can be controlled by the present invention, provided that the promoter operably linked to the coding sequence is genetically engineered to be complementary to the DNA binding domain of the USP receptor and, optionally, Lt; RTI ID = 0.0 > and / or < / RTI > For example, a target polypeptide useful for regulating plant fertilization is activated by a USP receptor polypeptide in the presence of a larval hormone or agonist thereof.

Also included in the invention are mutants of USP receptor polypeptides. Mutants are engineered to have a reduced level of background activation properties of the target expression cassette, leading to relatively greater induction than non-inferior rear expression. In addition, mutants can be developed in which their binding to the larval hormone or agonist thereof is modified. Mutants with modified binding properties will react with other agonists in a manner unique to these agonists. For example, the development of mutant USP receptors that react only with the non-hydroprenic agonist phenoxycabe distinguishes isoprenoid and non-isoprenoid haptenic hormone agonists. Useful mutagenesis methods such as chemical mutagenesis or specific mutagenesis are known in the art.

In yet another method, a mutant receptor polypeptide is produced by PCR mutagenesis of a nucleotide sequence encoding a ligand binding domain of USP. These mutant receptor polypeptides are expressed in host organisms which are themselves provided with convenient screening and separation techniques such as yeast. However, screening for mutant receptor polypeptides that exhibit reduced basal activity and greater pulp induction in such host organisms may provide only candidates for further testing in plant cells, which may result in a reduction in the amount of steroid and thyroid hormone Although receptors may act in yeast, they become apparent in studies using the glucocorticoid receptor (GR), which can not predict functionality in transgenic plants (Lloyd et al., Science 226: 436 (1994)). A limitation in applying results from yeast is that yeast cells expressing GR do not react with the commonly used chemical ligand dexamethasone, but this ligand acts in other heterogeneous systems (Schena et al., Proc. Natl. Acad. Sci. USA 88: 10421-10425 (1991)).

Another test in plant cells is accomplished by constructing receptor expression cassettes that encode mutated receptor polypeptides and transforming them into plant cells using a target expression cassette. Transformed plant cells are tested for the activation of the 5'-regulated region of the target expression cassette by a mutant receptor polypeptide in the presence of the nematicide or agonist thereof. Mutant receptor polypeptides that induce high expression of the target polypeptide in the presence of a low basal expression of the target polypeptide and a larval hormone or agonist thereof in the presence of the insect hormone or agonist thereof are useful for controlling gene expression in plants.

As described above, the method of the present invention can be used to statistically increase gene expression beyond a minimum basal level. However, the present invention can be used to statistically reduce or inhibit activation of gene expression mediated by a complex formed by a receptor, such as USP and EcR. The modulation of gene expression in plants mediated by such receptor complexes is an object of PCT / EP 96/00686, filed March 3, 1995, which is incorporated herein by reference. Reversal of activation mediated by these receptor complexes is triggered by the presence of the larval hormone or agonist thereof, which is a chemical ligand for USP receptor polypeptides (see FIGS. 2 and 3). In the presence of a larval hormone or agonist thereof, the USP: EcR complex is cleaved, thereby reversing activation. For example, in a transgenic plant that expresses a USP and GAL4-EcR-Cl receptor polypeptide and contains a target expression cassette with a GAL4 binding site element, the modulation of gene expression of the target polypeptide induced by the complex will be reversed. This reversal can occur in the presence of tebufenozide (also known as RH 5992), or other chemical ligands that bind to the ligand binding domain of EcR, or in the absence of such chemical ligands.

For expression in plants, suitable promoters must be selected for both the receptor expression cassette and the target expression cassette. Unless otherwise specifically stated, the promoters mentioned below can be used to induce the expression of a receptor polypeptide or a target polypeptide in a plant. These promoters include, but are not limited to, constitutive, inducible, transiently regulated, developmentally regulated, chemically regulated, tissue-preferred and tissue-specific promoters. Preferred constitutive promoters include, but are not limited to, the CaMV 35S and 19S promoters [US Pat. No. 5,352,605]. Additional preferred promoters include, but are not limited to, some of the actin genes known to be expressed in most cell types. McElroy et al., Mol. Gen. Genet. 231: 150-160 (1991) can easily be incorporated into the receptor expression cassettes of the present invention and are suitable for use in monocot plant hosts. Another preferred constitutive promoter is derived from ubiquitin, another gene product known to accumulate in a number of cell types. The ubiquitin promoter is cloned from several species for use in transgenic plants (see Sunflower-Binet et al., Plant Science 79: 87-94 (1991); Corn-Christensen et al., Plant Molec. Biol. 12: 619-632 (1989)). The maize ubiquitin promoter has been developed in transgenic monocotyledonous plants and the sequences and vectors thereof constructed for transformation of monocotyledonous plants are described in EP-A-342 926. The ubiquitin promoter is suitable for use in the present invention in transgenic plants, especially monocotyledons. Additional useful promoters include U2 and U5 snRNA promoters from maize (Brown et al., Nucleic Acids Res. 17: 8991 (1989)) and promoters from alcohol dehydrogenase (Dennis et al., Nucleic Acids Res. 12: 3983 (1984).

A tissue-specific or tissue-preferred promoter useful in the present invention for plants, particularly corn, is a promoter that induces expression in roots, sponges, leaves or pollen. Such promoters are described in WO 93/07278, the contents of which are incorporated herein by reference. A promoter that provides seed-specific expression (Schernthaner et al., EMBO J. 7: 1249 (1988), the contents of which are incorporated herein by reference), EP-A- Specific promoters ant32 and ant43D, the drug (tappet) -specific promoter B6 (Huffman et al., J. Cell. Biochem. 17B: Abstract # D209 (1993)]; A pistil-specific promoter such as the modified S13 promoter (Dzelkalns et al., Plant Cell 5: 855 (1993)) is useful.

In addition, chemically-induced promoters are useful in the present invention. Certain promoters of this method useful for inducing the expression of receptor polypeptides or target polypeptides in plants are described, for example, in EP-A-332 104, the contents of which are incorporated herein by reference.

In addition, the 5 ' regulatory region of the receptor expression cassette or target expression cassette may comprise other enhancer sequences. A number of sequences have been found to enhance gene expression in transgenic plants. For example, it has been found that a number of non-detoxified leader sequences derived from viruses enhance expression. Specifically, it has been reported that tobacco mosaic virus (TMV, "Ω-sequence"), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) ≪ / RTI > were found to be effective in enhancing expression (Gallie et al. Nucl. Acids Res. 15: 8693-8711 (1987); Skuzeski et al. Plant Molec. Biol. 15: 65-79 (1990). Other leader sequences known in the art include, but are not limited to, the following leader sequences:

* Picornavirus readers, eg EMCV readers (Encephalomio Cardidis 5 'unencrypted domain) (Elroy-Stein, O., Fuerst, TR, and Moss, 6126-6130 (1989);

* Potyvirus readers such as the Tobacco Etch Virus (Allison et al., (1986); MDMV reader (corn dwarf mosaic virus); Virology, 154: 9-20;

Human immunoglobulin heavy chain binding protein (BiP) leader (Macejak, D. G., and Sarnow, P., Nature, 353: 90-94 (1991);

A comparative leader from the envelope protein mRNA of the Arabidopsis mosaic virus (AMV RNA 4) (Jobling, SA, and Gehrke, L., Nature, 325: 622-625 (1987));

* Tobacco Mosaic Virus Reader (TMV) (Gallie, D.R. et al., Molecular Biology of RNA, pages 237-256 (1989)] and

* Corn Chlorotic Stain Virus Reader (MCMV) (Lommel, S.A. et al., Virology, 81: 382-385 (1991), Della-Cioppa et al., Plant Physiology, 84: 965-968 (1987)].

Various intron sequences have been found to enhance expression, particularly when added to the 5 ' regulatory region in terminal cells. For example, the maize Adh1 gene has been found to significantly enhance the expression of wild-type genes under homologous promoters when introduced into maize cells (Callis et al., Genes Develep 1: 1183-1200 (1987)).

In addition to introducing one or more of the aforementioned elements into the 5 'regulatory region of the target expression cassette, other elements specific to the target expression cassette may also be introduced. Such factors include, but are not limited to, minimal promoters. By the minimal promoter, the base element is highly inactivated or almost inactivated without binding the site for the upstream activator. Such promoters have low rear activity in plants in the absence of transactivators or in the absence of enhancers or reactive element binding sites. Any minimal promoter particularly useful for the target gene in plants is the Bzl minimal promoter obtained from the bronze1 gene of maize. The Bzl core promoter is obtained from the 'myc' mutant Bzl-Luciferase construct pBzlLucR98 by cleavage at the Nhel site located at -53 to -58 (Roth et al., Plant Cell 3: 317 (1991) . The Bzl core promoter fragment thus derived extends from -53 to +227 and contains Bzl intron-1 in the 5 ' -readed region when used in transgenic maize.

In addition to promoters, various 3 'transcription terminators can be used in the present invention. Transcriptional terminators contribute to the termination of transcription and correct mRNA polyadenylation. Suitable transcription terminators and transcription terminators known to act on plants include CaMV 35S terminator, tml terminator, nopaline synthetase terminator, pea rbcS E9 terminator, and other terminators known in the art. They can be used for both monocotyledonous and dicotyledonous plants.

The expression cassettes of the present invention can be introduced into plant cells in a number of ways recognized in the art. Those of ordinary skill in the art will appreciate that the method may be selected according to the plant type that is the target of transformation, that is, monocotyledonous or dicotyledonous plants. Suitable methods of transforming plant cells include, but are not limited to, microinjection (Crossway et al., BioTechniques 4: 320-334 (1986)), electroporation (Riggs et al., Proc. Natl. Acad. Sci. USA 83: 5602-5606 (1986), Agtobacterium-mediated transformation (Hinchee et al., Biotechnology 6: 915-921 (1988)), direct gene transfer (Paszkowski et al., EMBO J. 3: 2717-2722 (1984); and ballistic particle accelerations using devices available from Biologicals of Agrace, Inc., Hercules, Calif., Madison, Wis. (Sanford et al., U.S. Patent No. 4,945,050; And McCabe et al., Biotechnology 6: 923-926 (1988)]. Other references include: Weissinger et al., Annual Rev. Genet. 22: 421-477 (1988); Sanford et al McCabe et al., Bio / Technology 6: 923- (1987) (onion), Plant Physiol. 87: 671-674 (1990) (rice); Klein et al., Proc. Natl. Acad. Sci. USA, 85: 4305-4309 (1988) (Datta et al., Bio / Technology 8: 736-740 (maize); Klein et al., Bio / Technology 6: 559-563 (1988) Klein et al., Plant Physiol. 91: 440-444 (1988) (maize); Fromm et al., Bio / Technology 8: 833-839 (1990) (maize); and Gordon-Kamm et al. , Plant Cell 2: 603-618 (1990) (maize); Svab et al., Proc. Natl Acad. Sci. USA 87: 8526-8530 (1990) (tabacco chloroplast); Koziel et al., Biotechnology 11: 194-200 (1993) (Maize); Shimamoto et al., Nature 338: 274-277 (1989) (rice); Christou et al., Biotechnology 9: 957-962 (1991) (rice); EP-A-332 581 (orchardgrass and other Pooideae); Vasil et al., Biotechnology 11: 1553-1558 (1993) (wheat); Weeks et al., Plant Physiol. 102: 1077-1084 (1993) (wheat).

A particularly preferred embodiment for introducing the expression cassette of the present invention into corn by microinjection shock is described in Koziel et al, Bio / Technology 11: 194-200, 1993, the contents of which are incorporated herein by reference Quot; Another preferred embodiment is a protoplast transformation method for maize as described in EP-A-292 435, which is incorporated herein by reference. Particularly preferred embodiments for introducing the expression cassette of the invention into the mill by microinjection shock are described in WO 94/13822, which is hereby incorporated by reference in its entirety.

Transformation of plants can be carried out using single DNA molecules or multiple DNA molecules (i.e., co-transformation), both of which are suitable for use in the expression cassettes of the present invention. A number of transformation vectors can be used for plant transformation, and the expression cassettes of the present invention can be used in combination with all such vectors. The choice of vector will depend on the desired transformation technique and the target species for transformation.

Many vectors can be used for transformation using Agrobacterium tumefaciens. These typically include one or more T-DNA, and include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)]. In one preferred embodiment, the expression cassettes of the invention can be inserted into binary vectors pCIB200 and pCIB2001 for use in Agrobacterium. These vector cassettes for Agrobacterium-mediated transformation are constructed in the following manner. pTJS75kan was prepared by Narl digestion of pTJS75 (Schmidhauser & Helinski, J Bacteriol. 164: 446-455 (1985)], the tetracycline-resistant gene is cut and the Accl fragment from pUC4K containing NPTII is inserted (Messing & Vierra, Gene 19: 259-268 (1982); Bevan et al., Nature 304: 184-187 (1983); McBride et al., Plant Molecular Biology 14: 266-276 (1990)]. The Xhol linker was ligated to the EcoRV fragment of pCIB7 containing left and right T-DNA borders, a plant selectable nos / nptll chimeric gene and a pUC polylinker (Rothstein et al., Gene 53: 153-161 (1987)], the Xhol-digested fragment was cloned into SaII-digested pTJS75kan to construct pCIB200 (see EP-A-332 104, Example 19). pCIB200 contains the following unique polylinker restriction sites: EcoRI, Sstl, Kpnl, BgIII, Xbal and Sall. Plasmid pCIB2001 is a derivative of pCIB200 inserted into another restriction site polylinker. The unique restriction sites in the pCIB2001 polylinker are EcoRI, Sstl, Kpnl, Bglll, Xbal, Sall, Mlul, Bcll, Avrll, Apal, Hpal and Stul. In addition to containing these unique restriction sites, pCIB2001 contains the plant and bacterial Kanamycin selection, left and right T-DNA borders for Agrobacterium-mediated transformation, RK2-induced trfA function for immobilization between cola and other host, and OriT and OriV functions from RK2. The pCIB2001 polylinker is suitable for cloning plant expression cassettes containing their own regulatory signal.

Another vector useful for Agrobacterium-mediated transformation is the binary vector pCIB10, a gene encoding kanamycin resistance for selection in plants, T-DNA right and left border sequences, Lt; RTI ID = 0.0 > pRK252 < / RTI > that allows replication in both E. coli and Agrobacterium. Its construction method is described in Rothstein et al., Gene 53: 153-161 (1987). Various derivatives of pCIB10 have been constructed to introduce genes for the hygromycin B phosphotransferase described in Gritz et al., Gene 25: 179-188 (1983). These derivatives enable the selection of transgenic plant cells on hygromycin alone (pCIB743), or hygromycin and kanamycin (pCIB715, pCIB717).

Methods employing direct gene transfer or Agrobacterium-mediated delivery typically involve the use of a variety of immunomodulators such as, but not limited to, resistance to antibiotics (e.g., kanamycin, hygromycin or methotrexate) or herbicides such as phosphinotricin Lt; RTI ID = 0.0 > selectable < / RTI > However, the selection of selectable markers for transformation of plants is not important to the present invention.

In the case of certain plant species, different antibiotics or screening markers of the manufacturer may be preferred. A selectable marker commonly used for transformation is the nptll gene (see Messing & Vierra, Gene 19: 259-268 (1982)), which provides resistance to kanamycin and related antibiotics; Bevan et al., Nature 304: 184-187 (1983)], the bar gene providing resistance to the herbicide phosphinotricin [White et al., Nucl Acids Res 18: 1062 (1990), Spencer et al , Theor Appl Genet 79: 625-631 (1990)], resistance to the hph gene providing resistance to the antibiotic hygromycin (Blochinger & Diggelmann, Mol Cell Biol 4: 2929-2931) and methotrexate Dhfr gene (Bourouis et al., EMBO J. 2: 1099-1104 (1983)).

One such vector useful for direct gene transfer technology with screening by the herbicide Basta (or phosphinotricin) is pCIB3064. This vector is based on the plasmid pCIB246, It contains CaMV 35S at operational fusion to the E. coli GUS gene and the CaMV 35S transcriptional terminator and is described in WO 93/07278 incorporated herein by reference. One gene that provides resistance to phosphinotricin is the bar gene from Streptomyces viridochromogenes (Thompson et al., EMBO J 6: 2519-2523 (1987)). This vector is suitable for cloning plant expression cassettes containing their own regulatory genes.

Another transformation vector is a selectable marker that provides resistance to methotrexate. PSOG35 using the collagen gene dihydrofolate reductase (DHFR). PCR is used to amplify the intron 6 from the 35S promoter (approximately 800bp), the maize Adh1 gene (~550bp) and the 18bp of the leader sequence compared to the GUS from pSOG10. this. The 250 bp fragment encoding the collidine dihydrofolate reductase type II gene was also amplified by PCR and ligated with Sacl-Pstl fragments from pB1221 (Clontech) containing the pUC19 vector backbone and nopaline synthase terminator to generate these two Assemble the PCR fragment. Assembly of these fragments yields pSOG19 containing the 35S promoter upon fusion with the intron 6 sequence, the GUS leader, the DHFR gene and the nopaline synthase terminator. Replacing the GUS leader in pSOG19 with the leader sequence from corn chlorotic staining virus assay (MCMV) generates the vector pSOG35. pSOG19 and pSOG35 contain pUC-derived genes for ampicillin resistance and have HindIII, SphI, PstI and EcoRI sites available for cloning of foreign sequences.

One of the advantageous aspects of the present invention is that it is used to control plant fertilization under packaging conditions. Effective fertilization can be measured as the percentage of seeds that induce the formation of viable conglomerates and form viable conglomerates. Modifications according to the invention can be modulated by introducing a nucleotide sequence encoding a suitable target into the target expression cassette wherein expression of the target polypeptide interferes with plant modification to statistically reduce or increase plant modification. In a preferred embodiment of the present invention, the target polypeptide makes the modification process ineffective, which means that the formation of the viable conjugate will be prevented or inhibited. Such ineffective fertilization can be measured as a percentage of seeds that form viable conglomerates and can be induced by various means. This includes, but is not limited to, 1) destruction or deformation of these processes that are important for the formation of viable conglomerates, 2) incapacitating pollen or escape, if formed, or 3) backpacks, pistils, Incorrect development of the tract is included. In the present invention, the larval hormone or agonist thereof is applied to the transplanted plant or in contact with the plant under packaging conditions, which activates the expression of the target polypeptide, rendering the fertilization impotent. In another embodiment of the present invention, the expression of the target polypeptide restores plant fertility.

It will be appreciated that a different degree of effective or ineffective modification can be achieved by the present invention. In a preferred embodiment, an ineffective correction of 80% or more and more preferably 95% or more can be achieved. The ability to provide differences in modification levels makes the invention suitable for a variety of agricultural purposes.

Useful coding sequences for the target polypeptides include, but are not limited to, all sequences that encode the product that can disable the modification. These coding sequences may be homologous or heterologous in origin. The gene products of these coding sequences include, but are not limited to:

Diphtheria toxin A-chain (DTA) inhibiting protein synthesis (Greenfield et al., Proc. Natl. Acad., Sci .: USA, 80: 6853 (1983); Palmiter et al., Cell, 50: 435 (1987);

Pectate lyase peIE from Erwinia chrysanthemi EC16 (Keen et al., J. Bacteriology, 168: 595 (1986)) which degrades pectin causing cell lysis;

* T-urf13 (TURF-13) from the cms-T maize mitochondrial genome (this gene encodes a polypeptide designated URF13, which breaks down mitochondria or plasma members) (Braun et al., Plant Cell, 153 (1990); Dewey et al., Proc. Natl. Acad. Sci .: USA, 84: 5374 (1987); Dewey et al., Cell, 44: 439 (1986)];

Beta-1, 3 glucanase (Worral et al., Plant Cell 4: 759-771 (1992)), which causes premature degradation of the sporozoic callus wall;

Gin recombinase from the phage Mu a gene encoding a specific adenoviral recombinase that results in genomic rearrangement and loss of cell viability when expressed in plant cells (Maeser et al., Mol. Gen. Genet., 230: 170-176 (1991);

* Indoleacetic acid-lysine synthase (iaaL) from Pseudomonas syringae encoding an enzyme that binds lysine to indoleacetic acid (IAA) (when expressed in plant cells, (See, for example, Romano et al., Genes and Development, 5: 438-446 (1991); Spena et al., Mol. Gen. Genet., 227: 205-212 (1991); Roberto et al., Proc. Natl. Acad. Sci .: USA, 87: 5795-5801);

* Ribonuclease from Bacillus amyloliquefaciens (see Mariani et al., Nature 347: 737-7), which is known as a vanase that degrades mRNA in expressed cells, inducing cell death, 741 (1990); Mariani et al., Nature 357: 384-387 (1992)] and

* CytA toxin from Bacillus thuringiensis israeliensis, which encodes a mosquito-killing and hemolytic protein, which, when expressed in plant cells, leads to apoptosis due to the destruction of the cell membrane [cf. : McLean et al., J. Bacteriology, 169: 1017-1023 (1987); Eller et al., United States Patent No. 4,918,006 (1990)].

Such polypeptides also include adenine phosphoribosyltransferase (APRT) (Moffatt and Somerville, Plant Physiol., 86: 1150-1154 (1988)); DNAse, RNAse; Protease; Salicylate hydroxylase, and the like.

It is clear that the target expression cassette may comprise a 5 ' regulatory region operably linked to a nucleotide sequence important for the formation of a viable conformation such as APRT when transcribed. On the other hand, ribozymes can be used to target mRNA from genes important for the formation of the zygote. Such a ribozyme may comprise at least a portion of the target RNA and a hybridization region of about nine nucleotides complementary to the nucleotide sequence and a catalytic region suitable for cleaving the target RNA. Ribozymes are described in EP-A-321 201 and WO 88/04300 incorporated herein by reference (Haseloff and Gerlach, Nature, 334: 585-591 (1988); Fedor and Uhlenbeck, Proc. Natl. Acad. Sci .: USA, 87: 1668-1672 (1990); Cech and Bass, Ann. Rev. Biochem., 55: 599-629 (1986); Cech, T. R., 236: 1532-1539 (1987); Cech, T.R. Gene, 73: 259-271 (1988); and, Zang and Cech, Science, 231: 470-475 (1986)].

In addition, the nucleotide sequence encoding the target polypeptide may be operably linked to a 5 ' regulatory sequence that induces its expression in a tissue- or cell-specific manner. Means for providing such tissue- or cell-specific expression have been described above. Such expression specificity will only show the effect of the target polypeptide in the tissues or cells necessary for the formation of viable conglomerates and will not have deleterious effects on the plant beyond the effect on fertilization.

Within the scope of the present invention, it is recognized that male fertilization of transgenic plants, magnetic fertilization of transgenic plants, or both can be regulated. Male sterility can cause defects that lead to pollen non-formation or lack of functionality in pollen if it is formed. Thus, when the pollen is not formed or is formed, it is impossible to survive or undergo effective modification under normal conditions.

Magnetic infertility is the inability or inability to produce functional or viable giant spores or backpacks, or other tissues, required for pollen germination, development or fertilization. Magnetic infertility will result in defects leading to non-formation of giant spores or backpacks, or inadequate development of ovaries, ovules, pistils, beakers, or infiltration tubes. Thus, a viable backpack is not developed, and if it is formed, effective modification under normal conditions is impossible.

For example, transgenic plants can be obtained that express USP receptor polypeptides or polypeptides in a drug using a drug-specific promoter operably linked to a suitable nucleotide sequence. In addition, the transgenic plant further comprises a target expression cassette having a 5 'regulatory sequence comprising a suitable response element sequence together with a core promoter element from Bzl operably linked to an encoding sequence for a ribonuclease vanname something to do. Upon application of a larval hormone or agonist thereof to a transgenic plant that expresses a USP receptor polypeptide, activation of the 5 ' regulatory sequence of the target expression cassette occurs with subsequent production of the target polypeptide canase. Expression of drug-specific vanazases leads to apoptosis and consequently male sterility. Magnetic infertility can be generated with similar combinations of receptor polypeptides and target expression cassettes using a pistil-specific promoter operably linked to a nucleotide sequence encoding a receptor polypeptide.

On the other hand, plants can be genetically engineered, in which the expression of the target polypeptide restores virility to male-infertile or magnetic-infertile plants. For example, the vancomycin gene may be expressed under the control of the Ant43D, Ant32 or B6 promoter, or may be transfected with the TA29 promoter under the control of Mariani et al., Nature 347: 737-741 (1990) and Mariani et al. Nature 357: 384-387 (1992). These plants further include a receptor expression cassette for any second receptor polypeptide from a constitutive promoter such as the USP receptor polypeptide and the same drug-specific promoter or maize ubiquitin, 35S or rice actin promoter. These plants also contain a target expression cassette having a 5 'regulatory sequence comprising a suitable response element with a core promoter element from Bzl operably linked to the coding sequence for the vanadate inhibitor Varasta, Upon application of its agonists, activation of the 5 'regulatory sequence of the target expression cassette occurs with subsequent production of the target polypeptide Varasta. Varasta inhibits ribonuclease activity of vanazines and promotes drug and pollen development normally. Whereby the correction is restored.

Similar approaches can be used to control magnetic infertility. Magnetic-infertile plants can be obtained by using promoters specific for expression in magnetic reproductive tissues instead of drug-specific promoters for inducing vanadate expression. Magnetic modifications are restored by the introduction of a target expression cassette containing a Varasta coding sequence by the larval hormone or its agonists.

This approach can use all magnetic- or male-infertile genes and can construct recovery genes. An efficacious recovery gene other than Varasta is described in EP-A-412 991.

The genetic traits are genetically engineered into the transgenic plants described above and their seeds are delivered by male reproductive or asexually proliferative growth and thus can be maintained and propagated to offspring plants. In general, such maintenance and breeding makes the use of the developed known agricultural methods suitable for certain purposes such as cultivation, sowing or harvesting. Certain methods such as hydroponic cultivation or greenhouse cultivation techniques may also be applied. Since crops are susceptible to competition for weeds as well as attacks and damages caused by insects and infections, we take measures to control weeds, plant diseases, insects, nematodes and other inverted conditions to improve production. This includes the application of pesticides such as soil tillage or mechanical means such as the removal of weeds and infested plants, as well as herbicides, spouse sterile organic chemicals, nematicides, growth regulators, aging agents and insecticides.

The use of the beneficial genetic properties of transgenic plants and seeds according to the present invention further provides an improvement such as resistance to plague or stress, improved nutritional value, increased yield, improved structure to reduce losses due to lapping or shattering The present invention can provide a plant breeding aimed at the development of a plant having the characteristics as described above. The various stages of breeding are characterized by clear human intervention, such as screening of hybridized populations, induction of moisture in crossbreeding populations, or selection of appropriate offspring. Different breeding methods are selected depending on the desired characteristics. Related arts are known in the art and include, but are not limited to, crossing, inbreeding, cross breeding, cross breeding, cross breeding, interspecific crossing, and islet technique. Thus, the transgenic plants and their seeds according to the present invention may be used in plants for the treatment of diseases such as, for example, improved botanicals such as herbicides or insecticide treatment, which increase the effectiveness of conventional methods, It can be used for breeding. On the other hand, it is possible to obtain new crops with improved stress tolerance, yielding yields of better quality than products which can not withstand comparable reverse development conditions due to optimized genetic " devices ".

Germination characteristics and uniformity of seed harvested and sold by growers are not important, while germination characteristics and uniformity of seeds in seed production are essential production characteristics. Because it is difficult to maintain seeds that do not contain other crops and weed seeds and to relieve seed infectious diseases and produce seeds with good germination, a fairly wide and clear seed production run has been developed by seed producers, And sales. It is therefore common practice for growers to purchase certified seeds that meet certain quality specifications instead of using harvested seeds from their own crops. The vigorous plants used as seeds can be conventionally treated with protective coatings comprising herbicides, insecticides, fungicides, bactericides, nematocides, molluscs or mixtures thereof. Conventional protected coating agent used include mercaptans, car duplex, tiram (TMTD ^R), metal raksil (Apron ^R), and the pyrimidine force - are included, such as methyl (Actellic ^R) compound. Optionally, these compounds are formulated with additional carriers, surfactants or application-promoting adjuvants conventionally used in the field of formulation to protect against damage caused by bacteria, fungi or animal pests. The protective coating may be applied by immersing the plant in the liquid formulation or by coating it with a wet or dry formulation formulated. In addition, other application methods such as eye or fruit induced treatment may be used.

In addition, the present invention provides a novel agricultural method such as the above-exemplified method, characterized by using a transgenic plant, transgenic plant material, or transplanted seed according to the present invention.

The present invention can be used for all plants that can be transfected and regenerated with transgenic plants. Male sterility, magnetic sterility, or both, by the application of suitable chemical ligands. Control of plant fertilization is particularly useful for the generation of hybrid seeds. In order to produce hybrid seeds that are not contaminated with self-fertilized seeds, moisture control methods must be implemented to ensure hygroscopic-moisture rather than self-moisture. This is typically accomplished by mechanical, genetic or chemical hybridization (CHA). For example, the current practice for corn is a mechanical abstraction method of magnetic (or seed) breeding, a time consuming and labor intensive method. In wheat, the control of fertilization by mechanical means can not be carried out on a seed production scale, and the genetic source of fertility control has not been established. The use of the present invention in producing hybrid seeds provides the reliability, ease of use and control benefits of male or female fertilization.

Transgenic plants containing appropriate receptor expression cassettes and target expression cassettes can be homozygous and can be maintained indefinitely. To obtain hybrid seeds, the homozygous lines of hybrid 1 and hybrid 2 are hybridized. As an example of using the present invention to produce hybrid seeds, crossed parent 1 is genetically engineered to be male sterile in the presence of a larval hormone or agonist thereof. By contacting both crossed 1 and crossed 2 with a larval hormone or agonist thereof, only successful seed production can yield crossed 2 pollen that corrects the crossed crosses. As a second embodiment of the present invention, it is possible to genetically engineer transgenes 1 to make them male-sterile in the presence of a transgender hormone or agonist thereof, genetically engineer transgenes 2 to detect the presence of a transgene hormone or agonist thereof To make them magnetic infertility. Each strain is maintained by self-fertilization by contacting the hybrid 1 and hybrid 2 with the larval hormone or its agonist. To produce hybrid seeds, two hybrid lines are interfused and only hybrid seeds are obtained. The fertilization is restored to the offspring hybrid plants by the introduced recovery genes. By these means, all desired hybrid seeds can be produced.

The chemical control of plant gene transplantation expression modulates vigorous developmental changes in target crops, changes crop plants to better suit the adverse environment, changes the flow of specific metabolic pathways, or induces high expression of the desired single protein product . ≪ / RTI > Chemical control of developmental programs in plants can direct growers when specific events such as flower development, foliage or fruit excretion, or other important developmental conditions are initiated. This chemical control of a specific developmental event not only gives farmers greater flexibility during transplantation and harvesting, but also allows them to respond to specific environmental conditions, such as early winter or wet spring. Such changes can be accomplished by modulating genes important for growth or regulatory pathways, such as the dissociated genes of Drosophila.

It may also be useful in regulating gene expression in the metabolic pathway. Plants can consume only a fixed amount of energy and all of the enhancement of the biosynthesis of a particular pathway, e. G., Storage proteins, leads to simultaneous loss of biosynthetic flow through the synthesis of energy-competitive pathways, e. . If such a change in biosynthetic pathway is allowed only after the plant has completed a particular developmental stage (i.e., maturation to growth), chemical control of gene expression may be useful or required for the particular biosynthetic change desired to be achieved .

Chemical modulation of gene expression may be useful for highly overexpressing specific proteins. Certain proteins are known to be toxic to a cell when expressed in a heterologous host, in an exogenous < RTI ID = 0.0 > complement < / RTI > complement, or when overexpressed. Chemically modulating such proteins may be beneficial to normal plant growth or may be required to obtain sufficient amounts of plants to use plants for biosynthetic protein factors. Proteins that may be useful for large-scale biosynthesis in plants include industrial enzymes, pharmaceutical proteins, antigens, and other proteins.

Biopsies for identifying ligands for steroid hormone receptors are known (Evans et al., U.S. Pat. No. 5,298,429). The described receptors are restricted to glucocorticoids, mineralocorticoids, estrogen-related and thyroid hormone receptors. These receptors are transformed into murine kidney cell cultures (CV-1 or COS cell lines) and tested for their ability to transactivate the expression of chemical CAT genes in the presence of suitable mammalian hormones. No receptor has been described other than transgenic plant cells with these receptors, the construction of plant-expressible genes, and receptors with mammalian hormones as ligands.

USP has been suggested to be an " insect retinoid receptor " (Oro and Evans, WO 91/14695). In a predictive example, XR2C encoding USP is transformed into an insect cell culture (S2 cell line) to transactivate the chimeric CAT gene in response to the addition of retinoic acid. This technical description suggests that insect or animal cells transformed with such " insect retinoid receptor " can be used to screen for compounds capable of inducing receptor activation. However, Oro et al. Later show that USP is not activated by retinoic acid in the drosophila cell-culture assay and that USP does not react with any of the retinoids or testosterone hormones containing methoprenic acid under conditions where RXR is reactive (Harmon et al. And hereby incorporated by reference).

According to our finding that the larvae hormone and its agonists are ligands to USP and that USP can be used to activate target gene expression in plant cells in the presence of a larval hormone or agonist thereof, Lt; RTI ID = 0.0 > ligand. ≪ / RTI > Screening is based on the expression of a target expression cassette encoding a receptor-regulated reporter gene in a transgenic plant or plant cell expressing a transgenic USP receptor polypeptide and an optional second receptor polypeptide different from the USP receptor polypeptide. For the ability of these to induce USP receptor-regulated activation of the target polypeptide expression, the chemicals to be tested are placed in contact with the transplanted plant or plant cell in varying concentrations, and then a test for receptor gene expression is performed The expression of the target polypeptide is measured. A test substance showing an activation or a substantial increase in expression of the target polypeptide and a test substance showing inhibition or statistically significant decrease in the expression of the target polypeptide are identified as ligands of the USP receptor polypeptide. This method identifies a test substance that is not previously known as being a ligand for USP in the plant cell environment itself or identifies a test substance suspected to be a ligand of USP in the plant cell environment itself. Thus, the ligand of a USP receptor polypeptide can be produced by carrying out the following method according to the present invention:

Synthesizing a novel test substance according to a conventional method known in the chemical arts;

Transforming a plant cell with a USP receptor expression cassette encoding a USP receptor polypeptide and a target expression cassette encoding the target polypeptide;

Culturing the offspring cells of the transformed plant cells;

Expressing a USP receptor polypeptide in progeny cells;

Contacting the progeny cells with the synthesized test substance; And

Measuring the expression of the target polypeptide;

- repeating the previous two method steps with an additional novel test substance;

Selecting a test substance that significantly activates or inhibits the expression of the target polypeptide; And

- repeating the chemical synthesis of the selected material.

Ligands of USP receptor polypeptides that can be obtained according to the above method steps constitute another subject matter of the present invention.

Additionally, the methods of the invention can be used to identify antagonists or inhibitors of USP receptor-mediated activation of target polypeptide expression. Such antagonists are identified by their ability to reduce the ligand-induced activity of target polypeptide expression.

In the screening method, different reporter genes can be used as target expression cassettes. One useful reporter is firefly luciferase. Their use in a target expression cassette is described in Examples 7 and 9 below. Another useful reporter gene is GUS or a chromogenic substrate such as 5-bromo-4-chloro-3-indolyl beta-D-glucuronide or o-nitrophenyl- beta -D-glucuronide It is a glucuronidase that catalyzes cleavage. The GUS reporter has the advantage of producing a color reaction product which can be detected, for example, by quantitative analysis by spectroscopic analysis or qualitative analysis by visual inspection. Receptor expression cassettes useful for screening methods are USP, or chimeric variants of USP, such as USP-VP16 or VP16-USP.

Because USP is involved in the sequence with mammalian RXRa receptors, RXRs can form heterodimers with EcR (Thomas et al., Nature 362: 471-475 (1993)), receptor expression encoding RXR Cassettes can also be used for screening methods. In this way, a broad ligand for USP can be found to be useful as a ligand for RXR in plant cells, or a chemical other than a larval hormone or agonist thereof can be identified as a suitable chemical ligand for RXR in plant cells.

The following examples further illustrate the materials and methods used to practice the present invention and subsequent results. They are provided by way of illustration, and the details thereof are not to be construed as limiting the claimed invention.

Example 1: Construction of a plant-expressible receptor expression cassette encoding an exydon receptor

The DNA coding region for the Ecdysone Receptor (EcR) of Drosophila was amplified using a cDNA library derived from the Canton S pupa (6 days) prepared in lambda gt11 (Clontech, cat. no. IL 1005b], and fragments generated by genomic PCR using oligonucleotides designed from published sequences of the B1 isoform of EcR (Koelle et al., Cell 67: 59, 1991). The B1 isoform EcR sequence is confirmed by automated sequencing using standard methods and alignment with published sequences (Talbot et al., Cell 73: 1323, 1993). The entire length of the expressed EcR coding region was modified so that it was directly upstream of the start codon using the oligonucleotide SF43 (5'-CGC GGA TCC TAA ACA ATG AAG CGG CGC TGG TCG AAC AAC GGC-3 '; SEQ ID NO: 1) To contain the BamHI site. The plant expression vectors pMF6 and pMF7 contain the cauliflower mosaic virus 35S promoter (CaMV 35S), the maize Adhl intron 1, and the nopal cactus synthase polyadenylation and termination signals (Goff et al., Genes and Development 5: 298, 1991). The vectors pMF6 and pMF7 differ only in the orientation of the polylinker used for insertion of the desired coding sequence. The full length EcR coding sequence is linked to the plant CaMV 35S expression vector pMF6 by using a flanking BamHI restriction site. This receptor expression cassette is named 35S / EcR.

Example 2: Construction of a plant-expressible receptor expression cassette encoding an ultraspiracle receptor

CDNA encoding the pure Ultraspiracle receptor (USP) of Drosophila is described in Henrich et al., Nucleic Acids Research 18: 4143 (1990). The full-length USP coding sequence with flanking regions compared to flanking 5 'and 3' is ligated to the plant expression vector pMF7 (described in Example 1) using a flanking EcoRI restriction site. This receptor expression cassette is named 35S / USP.

Example 3: Construction of a receptor expression cassette comprising a DNA binding domain from GAL4 and a ligand binding domain from EcR

A receptor expression cassette is constructed in which the DNA binding domain of EcR is replaced with the DNA binding domain of GAL4 fused at the N-terminal position. The DNA coding region for EcR of Drosophila is obtained as described in Example 1. [ The coding sequence for the DNA binding domain of GAL4 is subcloned from the plasmid pMA210 (Ma and Ptashne, Cell, 48: 847 (1987)).

A receptor expressing cassette encoding the GAL4-EcR chimeric receptor polypeptide is constructed by fusing the DNA binding domain of GAL4 with the ligand binding domain of the EcR and the carboxy terminus. To perform fusion, oligonucleotides SF23 (5'-CGC GGG ATC CAT GCG GCC GGA ATG CGT CGT CCC G-3 '; SEQ ID NO: 2) were used to insert the same nucleotide positions as the amino acid residue 330 The BamHI site is introduced into the cDNA sequence by PCR. The resulting truncated EcR coding sequence (EcR ^330-878 ) is subcloned into the plasmid pKS + (Stratagene).

The coding sequence of the DNA binding domain, as described in Goff et al., Genes and Development 5: 298, 1991, by subcloning the amino terminus encoding the DNA sequence of GAL4 against ClaI into pSK + (Stratagene) (Amino acids 1 to 147) from the plasmid pMA210. This plasmid is designated pSKGAL2, digested with ClaI and KpnI, and then inserted into the following double-stranded oligonucleotide:

The obtained plasmid is named pSKGAL2.3. A fully fused 35S / GAL4-EcR ^330-878 is obtained using the BamHI site in the polylinker at both ends of the DNA binding domain of pSK + and the EcR ^330-878 residue of pKS +. These coding sequences are linked to the terminal lobe expression vector pMF6 (described in Example 1) using the flanking EcoRI restriction sites. This receptor expression cassette is designated 35S / GAL4-EcR ^330-878 .

Example 4: Construction of a plant-expressible receptor expression cassette comprising a ligand binding domain from ultraspiracle and a transactivation domain from VP16

A receptor expression cassette comprising the ligand binding domain of USP is constructed with the transactivation domain of VP16 fused to the N-terminal or C-terminal of the USP polypeptide.

In order to construct a receptor expression cassette encoding a chimeric polypeptide having the transactivation domain of VP16 at the C-terminal position, subcloning of pKS + (Stratagene) using the XhoI site in USP nucleotide number 1471 of the coding sequence resulted in the insertion of the receptor USP Terminal < / RTI > and stop codon of the cDNA for the target gene (described in Example 2). The resulting USP subclone encoding amino acids 1 to 490 is fused with the transactivation domain of VP16 using the flanking KpnI restriction site of the USP subclone and the KpnI site of pSJT1193CRF3 encoding the carboxy-terminal 80 amino acid of VP16 See: Triezenberg et al., Genes and Develop. 2: 718-729 (1988)]. The USP-VP16 fusion generated using the EcoRI and BamHI restriction enzymes located at both ends of the coding sequence of USP-VP16 is cloned into the CaMV 35S plant expression vector pMF7 (described in Example 1). This receptor expression cassette is designated 35S / USP-VP16.

The BamHI site adjacent to the USP start codon was first genetically engineered using the oligonucleotide SF42 (5'-CGC GGA TCC ATG GAC AAC TGC GAC CAG GAC-3 '; SEQ ID NO: 5) as a PCR reaction to generate an amino- USP < / RTI > The stop codon in VP16 was removed and a flanking BamHI site was introduced using oligonucleotide SF37 (5'-GCG GGA TCC CCC ACC GTA CTC GTC AAT TC-3 '; SEQ ID NO: 6) The start codon and BamHI site having the plant consensus sequence were amplified by PCR using oligonucleotide SA115 (5'-GTC GAG CTC TCG GAT CCT AAA ACA ATG GCC CCC CCG ACC GAT GTC-3 '; SEQ ID NO: 7) Lt; / RTI > The resulting VP16 activation domain and the USP coding sequence (encoding amino acids 1 to 507) were ligated in frame by an adjacent BamHI site and ligated into the CaMV 35S plant expression vector pMF7 by the 5'-BamHI and 3 ' -USP Encrypt sequence is inserted. This receptor expression cassette is named 35S / VP16-USP.

Example 5: Construction of a receptor expression cassette having a DNA binding domain and a ligand binding domain from EcoR and a transactivation domain from a C1 regulatory gene of maize

(5'-CGC-GGA-TCC-ATG-GGT-CGC-GAT-GAT-CTC-TCG-CCT-TC-3 '; SEQ ID NO: 8) used in the PCR reaction for the full length EcR- ^Lt ; RTI ID = 0.0 > ^EcR227-825- C1 < / RTI > fusion is obtained by substituting the start codon immediately preceding the EcR DNA binding domain. The coding sequence for the transcription activation domain of maize C1 protein (amino acids 219-273) (Goff et al. Genes and Develop. 5: 298-309 (1991)) is fused in frame with the coding sequence (at EcR KpnI restriction enzyme site) for amino acids 51 to 825 of EcR. The C1 transactivation domain is linked to EcR by polylinker (SEQ ID NO: 9) encoding VPGPPSRSRVSISLHA. The 35S / EcR ^227-825 -C1 plant expression vector fusion is constructed by inserting the BamHI fragment containing the coding sequence into the pMF7 vector. This receptor expression cassette is named 35S / EcR ^227-825 -C1.

Example 6: Construction of a receptor expression cassette having a DNA binding domain from GAL4, a ligand binding domain from EcR, and a transactivation domain from the C1 regulatory gene of maize

A GAL4-EcR ^330-825 -C1 fusion is constructed using the constructs GAL4-EcR ^330-878 described in Example 3 and the EcR ^227-825 -C1 construct of Example 5. The sequence of the EcR coding region (starting at amino acid 456) is exchanged at the AatII site. This receptor expressed cassette is named 35S / GAL4-EcR ^330-825- C1.

Example 7: Construction of a plant-expressible target expression cassette encoding a firefly luciferase with a response element to the GAL4 DNA binding domain

A plant-expressible target expression cassette encoding a firefly luciferase with a response element to the DNA binding domain of GAL4 is constructed in the following manner. The corn bronze-1 (Bzl) core promoter inducing the synthesis of firefly luciferase was removed from the Bzl reporter pBz1LucR98 (Roth et al., Plant Cell 3: 317, 1991) by the NheI and SphI sites and luciferase 0.0 > pUC6S-induced < / RTI > plasmid containing the gene. The modified Bzl core promoter contains the NheI site (GCTAGC) and a Bzl promoter sequence at nucleotide position -53 or less (Roth et al., Plant Cell 3: 317, 1991). 10 GAL4 binding sites were removed from the GAL4 regulated reporter pGALLuc2 by digestion with EcoRI and PstI (Goff et al., Genes and Development 5: 298, 1991), into pBlueScript (Stratagene) using the same restriction enzyme sites . The HindIII site at the 5 'end of the GAL4 binding site was changed to the BamHI site by insertion of the HindIII / BamHI / HindIII adapter and the resulting BamHI fragment containing the GAL4 binding site was removed to obtain the Luciferase derived Bzl core RTI ID = 0.0 > BglII < / RTI > upstream of the promoter. This target expression cassette is named (GAL4 _bs ) _10- Bz1 _TATA / Luc.

Example 8: Construction of a plant-expressible target expression cassette encoding a firefly luciferase with direct repeat response elements

A plant-expressible target expression cassette encoding the firefly luciferase with the direct repeat (DR) response element with the EcRE half-site and the RXR-preferred half site is constructed in the following manner: as described in Example 7 Similarly, the maize Bzl core promoter-luciferase construct in the pUC6S-induced plasmid is used as starting point. (SEQ ID NO: 10) (SF77: 5'-GAT CCG TAG GGG TCA CGA AGT TCA CTC GCA-3 '; SEQ ID NO: 10), which is a double-stranded synthetic oligonucleotide containing three base pairs between the half- : 5'-GAT CTG CGA GTG AAC TTC GTG ACC CCT ACG-3 '; SEQ ID NO: 11), phosphorylated and annealed, inserted into the unique BglII site and ligated upstream of the Bzl core promoter. Three copies of RE are obtained by sequential BglII digestion and insertion of additional double-stranded oligonucleotides. This target expression cassette is designated (DR3) _3- Bz1 _TATA / Luc.

Example 9: Transformation of plant cells and modulation of expression of a target polypeptide by a receptor polypeptide in the presence of a chemical ligand

The modulation of target polypeptide expression by various receptor polypeptides, including the chimeric receptor polypeptides of the present invention, can be accomplished by simultaneously transforming plant cells into the required gene constructs using high viscosity microinjection bombardment and performing biochemical assays for the presence of target polypeptides As shown in FIG. The necessary genetic constructs include a USP receptor expression cassette encoding the USP receptor polypeptide (FIG. 1). Optionally, the USP receptor expression cassette can be transformed with a second receptor expression cassette encoding USP and other receptor polypeptides (FIGS. 2 and 3). In addition, a target expression cassette encoding the target polypeptide is required.

Expression cassettes were prepared from corn suspension cultured in liquid N6 medium by high-viscosity microinjection shock using standard techniques of DNA precipitation with microinjection and high viscosity shock induced by compressed helium (PDS-1000 / He, BioRad, Hercules, CA) Cells (Chu et al. Scientia Sinica XVIII: 659-668, 1975]. Transfected cells are cultured in N6 medium for about 48 hours in the presence of a suitable chemical ligand in a liquid suspension. After incubation, the transformed cells are harvested and homogenized at 0 ° C. The fractions in the extract are removed by centrifugation at 10,000 g for 5 minutes at 4 ° C.

Target polypeptide expression is detected by analyzing the extract against the presence of the product encoded by the target expression cassette. When testing the expression control by a receptor polypeptide in the presence of a chemical ligand, the commonly used coding sequence for the target polypeptide is firefly luciferase. The activity of firefly luciferase is measured by quantifying the chemiluminescent produced by luciferase catalyzed phosphorylation of luciferin using ATP as a substrate using an Analytical Luminescence Model 2001 photometer.

Example 10: Receptor polypeptide GAL4-EcR ^330-825 -C1 and USP-VP16 activate the expression of the target polypeptide in plant cells, and this activation is blocked by the larval hormone agonist

Expression cassette 35S / GAL4-EcR ^330-825- C1 (Example 6), receptor expression cassette 35S / USP-VP16 (Example 4) and target expression cassette (GAL4 _bs ) _10- Bz1 _TATA / Luc (Example 7) is cotransformed into maize cells. The transformed cells are cultured for about 48 hours as a chemical ligand in the presence of 10 [mu] M phenoxycarb or methoprene. Luciferase assay is performed as described in Example 9. The results are shown in Table 1.

Receptor Chemical ligand Luciferase activity (optical unit) Experiment # 1 None None Phenoxycarbomethoprene 2,503277,86277,41814,786 No ^{35S / GAL4-EcR 330-825 -C1 +} 35S / USP-VP1635S / GAL4-EcR 330-825 -C1 + 35S / USP-VP1635S / GAL4-EcR 330-825 -C1 + 35S / USP-VP16 Experiment # 2 None None Metoprene 1,302178,0925,730 No 35S / GAL4-EcR ^330-825 -C1 + 35S / USP-VP1635S / GAL4-EcR ^330-825 -C1 + 35S / USP-VP16

The results show that the 5'regulatory region of the target expression cassette comprising the GAL4 response element can be activated in the plant cell by the receptor polypeptides GAL4-EcR-C1 and USP-VP16, and this activation can be activated in the presence of a lectin hormone agonist It is reversed. The expression level of the target polypeptide luciferase was 3.5 to 31 fold lower in the presence of the larval hormone agonist compared to in the absence of the larval hormone agonist.

Example 11 Receptor Polypeptide GAL4-EcR ^330-825 -C1 and USP-VP16 activate the expression of the target polypeptide in plant cells, and this activation is blocked by the larval hormone agonist

Expression cassette 35S / GAL4-EcR ^330-825- C1 (Example 6), receptor expression cassette 35S / USP-VP16 (Example 4) and target expression cassette (GAL4 _bs ) ₁₀ -Bz1 _TATA / Luc (Example 7) is co-transformed into maize cells. The transformed cells are cultured in the presence of 10 [mu] M tebufenozide for about 48 hours with or without 10 [mu] M phenoxycarb or methoprene as chemical ligands. Luciferase assay is performed as described in Example 9. The results are shown in Table 2.

Receptor Chemical ligand Luciferase activity (optical unit) none none 1,302 35S / GAL4-EcR ^330-825- C1 + 35S / USP-VP16 none 178,092 35S / GAL4-EcR ^330-825- C1 + 35S / USP-VP16 Tebufenozade 908,912 35S / GAL4-EcR ^330-825- C1 + 35S / USP-VP16 Tebufenozide + metoprene 159,873

The results show that the 5 'regulatory region of the target expression cassette comprising the GAL4 response element can be activated in the plant cell by the receptor polypeptides GAL4-EcR-C1 and USP-VP16 by reacting with the chemical ligand tebufenozide, Activation is blocked in the presence of the larvae hormone agonist methoprene. The level of tebufenozide-induced activation of luciferase gene expression increased approximately 6-fold when used alone and was completely blocked by the presence of the larval hormone agonist methoprene.

Example 12: Receptor polypeptide VP16-USP activates the expression of a target polypeptide in plant cells

Plasmids containing the receptor expression cassette 35S / VP16-USP (Example 4) and the target expression cassette (DR3) _3- Bz1 _TATA / Luc (Example 8) were transformed into maize cells using the transfection method of Example 9 And simultaneously transformed. The transformed cells are incubated for 48 hours in the presence of 10 [mu] M methoprene as a chemical ligand. Luciferase assay is performed as described in Example 9. The results are shown in Table 3.

Receptor Chemical ligand Luciferase activity (optical unit) None 35S / VP16-USP35S / VP16 None None Metoprene 5,69029,967485,458

The results show that the 5 ' regulatory region of the target expression cassette containing the direct repeat response element can be activated in plant cells in the presence of a receptor hormone agonist by the receptor polypeptide VP16-USP. The expression level of the target polypeptide luciferase was about 16-fold or more as compared to that observed in the absence of the larval hormone agonist.

Example 13: Construction of vectors for expressing EcR, USP or RXR derivatives and transforming Arabidopsis plants containing a receptor-regulated reporter

Agrobacterium T-DNA vector plasmids were constructed from the plasmids pGPTV-Kan and pGPTV-Hyg described above (Becker et al., Plant Mol. Biol. 20: 1195-1197 (1992). The SacI / HindIII uidA (GUS) reporter gene of both pGPTV-Kan and pGPTV-Hyg plasmids was replaced with a SacI / HindIII polylinker from pGEM4Zf (+), pSPORT1, pBluescriptKS (+), plC20H, or pUC18 to obtain plasmids pSGCFW, pSGCFX, pSGCFY, pSGCFZ, pSGCGA, pSGCGC, pSGCGD, pSGCGE, pSGCGF and pSGCGG are obtained. Subcloning of the 328 bp KpnI / HindIII with 10 GAL4 binding sites and the maize Bronze-1 TATA as described in Example 7 into the KpnI / HindIII site of the modified luciferase reporter plasmid pSPLuc + (Promega) yielded the plasmid pSGCFOl As a target expression cassette, a GAL4-regulated luciferase reporter is constructed as a T-DNA Agrobacterium plasmid. The 1.991 Kb KpnI / Xbal fragment from pSGCF01 containing the GAL4 binding site-Bz1 TATA-luciferase reporter was subcorted into the T-DNA vector by linking to the 7.194 NdeI / Spel fragment of pSGCFXl and the 4.111 NdeI / KpnI fragment of pSGCFZl Lt; / RTI > The resulting plasmid is designated pSGCGL1 and contains an NPTII selectable marker and a GAL4-regulated luciferase reporter induced by the nos promoter which provides resistance to kanamycin in transgenic plants. A GAL4-regulated GUS reporter with 10 GAL4 binding site, 35S TATA region and GUS coding region was constructed in the same manner and designated pAT86. A direct repetitive (DR) response element reporter with three copies of DRRE, Bz1 TATA, a luciferase coding region, and a nos terminator similar to that described in Example 8 was constructed in a manner similar to that described for pSGCGL1, and pSGCHU1 . Using the receptor expression cassettes described in Examples 3 to 6 above, a similar Agrobacterium T-DNA construct containing the CaMV 35S promoter and the nos polyadenylation signal is constructed. Nucleic acid and double stranded receptor constructs are prepared by subcloning appropriate expression cassettes into the GAL4-luciferase reporter pSGCGL1.

Example 14: Preparation of Arabidopsis gene expressing VP16-USP and containing DR-luciferase reporter

Arabidopsis thaliana (Columbia) is transformed into an Agrobacterium vector containing the CaMV 35S promoter and DR-luciferase reporter (described in Example 13 above) according to the following vacuum-infiltration process. Agrobacterium tumefaciens GV3101 while passing the in 2x YT medium at 28 ℃ for 24 to 30 hours to produce an electric competition GV3101 Agrobacterium cells by incubation with OD ₆₀₀ of 0.5 to 0.7 units. The cells are ice-cooled for 10-30 minutes and centrifuged at 5,000 rpm for 5 minutes at 4 < 0 > C. The supernatant is discarded and the cell pellet resuspended in 1 volume of ice-cold 10% glycerol. The cells are centrifuged again at 5,000 RPM for 5 minutes at < RTI ID = 0.0 > 4 C. < / RTI > The supernatant is discarded and the cell pellet resuspended in 0.05 volume of ice-cold 10% glycerol. The cells are centrifuged again at 5,000 RPM for 5 minutes at < RTI ID = 0.0 > 4 C. < / RTI > The supernatant is discarded and the cell pellet resuspended in 0.02 volumes of ice-cold 10% glycerol. The cells are centrifuged again at 5,000 RPM for 5 minutes at < RTI ID = 0.0 > 4 C. < / RTI > The supernatant is discarded and the cell pellet resuspended in 0.02 volumes of ice-cold 10% glycerol. The cells are centrifuged at 5,000 RPM for 5 minutes at 4 < 0 > C. The supernatant is discarded and the cell pellet resuspended in 0.01 volume of ice-cold 10% glycerol. The cells are aliquoted in an amount of 200 ml per 1.5 ml microfuge tube, rapidly frozen in liquid N ₂ and stored at -80 ° C. Electric competing cells are used 6 weeks prior to storage at -80 ° C. Frozen electric competing cells are thawed on ice and 40 ml are transferred to a precooled 1.5 ml microfuge tube. Add 1 ml of the appropriate Agrobacterium plasmid DNA (2 to 10 ng) to the thawed cells and mix on ice. The cell / plasmid mixture is transferred to a precooled 0.2 cm Bio-Rad electroporation cuvette and electroporated at 2.0 K volts, 600 Ohms, 25 μFarad with a time constant of 6 msec. Add 1 † ml of 2x YT medium to the electroporation cuvette, mix the cell / plasmid solution with the pipette tip, and transfer the contents to a fresh 1.5 ml microfuge tube. The cells are then incubated at 37 DEG C for 1 hour on a shaker at 200 RPM. The cells are centrifuged for 2 minutes with the Eppendorf microfuge speed set to 6, the supernatant is discarded and the cell pellet is resuspended in the remaining liquid. Resuspended cells are plated on LB medium plates containing appropriate antibiotics. The plates are incubated at 28-30 < 0 > C for 2-3 days. 50 ml of LB culture is inoculated into a single colony transformed in a 250 ml flask containing 100 μg / ml of rifampicin, 25 μg / ml of genomicin and 100 μg / ml of kanamycin. The cultures are incubated for 24 to 36 hours at 28 ° C at 250 RPM and 10 ml of culture are used to inoculate 500 ml LB + antibiotics in a 2 L flask. The cultures are incubated at 28 [deg.] C overnight with stirring at 250 RPM. Plasmid DNA is isolated from Agrobacterium culture and validated by restriction analysis.

Arabidopsis plants are grown in mesh-covered soil in a 3-in- ² plastic pot at 16 ° C for 16 hours and 8 hours for dark for 4 to 5 weeks at 20 ° C. The plant is grown until the fissured tissue is about 2 inches long. The floral fission tissue of the transfected Arabidopsis plants is removed two days before exposure to Agrobacterium. The Agrobacterium culture was centrifuged at 5,000 RPM for 5 minutes and the resulting pellet was resuspended in infiltrated medium (4.3 g MS salt / L, 5% sucrose, 0.01 mg / ml benzylaminurine, 100 ml / ml Silwet L77, pH 5.8) < / RTI > Arabidopsis plants are soaked in water to saturate the soil. 500 ml of the bacterial cell suspension is transferred to the lower part of the sterile vacuum desiccator, and the Arabidopsis plants in the port are placed on the upper and lower sides of the Agrobacterium solution. Vacuum is applied to the desiccator for 5 minutes and then slowly released. This vacuum treatment is repeated three times and the plants are washed with an excess of Agrobacterium and circulated to the growth chamber. Vacuum-infiltrated plants are matured and flowers are bloomed to obtain seeds. The resulting seeds are dried in a drying oven at 95 < 0 > C and low humidity for about 5 to 10 days. The seeds are ground and removed from the dried flower, followed by filtration through a 425 micron mesh sieve. It takes about 5 weeks to obtain the seed after vacuum infiltration. When completely dried, approximately 240 mg of seed is added to 1 ml of 70% EtOH, sterilized, completely vortexed and incubated at room temperature for 2 minutes. The seeds are centrifuged at high speed for a short time in Eppendorf microfuge, and the supernatant is removed. Pelleting the seeds a 1ml sterile buffer solution (part of 10% Triton X-1 10, 10 parts bleach, 20 parts dd H ₂ O) was resuspended in and incubated for 30 min at room temperature following which the cavity. The seeds are centrifuged at high speed for a short time in Eppendorf microfuge, and the supernatant is removed. Reproduce the seed in sterile dd H ₂ O 1ml was suspended, and centrifuged in a micro-vortex pyuji in that following a high speed, and the supernatant was removed. This washing step is repeated 3 times, then the seeds are transferred to a 50 ml centrifuge tube and finally washed in 5 ml dd H ₂ O. The seeds are centrifuged at full speed in a Beckman table top centrifuge for a short period of time. The supernatant was discarded and the seeds were resuspended in 24 ml of sterile 0.8 w / v% low melting point agarose at 50 占 폚, mixed and then mixed with a selection antibiotic (50 占 퐂 / ml of kanamycin or 50 占 퐂 / ml of hygromycin) (GM) plate (Murashige and Skoog, Physiol Plant 15: 473-479, 1962), each containing 3 ml of the culture medium, Plated seeds are incubated at 4 占 폚 for 24 hours in a dark state and then transferred to a growth chamber set at 20 占 폚 for 16 hours for light state and 8 hours for dark state. The germinated seeds are screened on the plate for 5-10 days, seedlings are transplanted into new sorting plates and then transferred to the soil for further screening after 5-10 days. The newly transplanted seedlings are covered with plastic wrap for 2-3 days and grown until the fissured tissue appears.

Example 15: Chemical induction of isolated transgenic plant tissue

Transgenic plants are tested for gene expression inducible by the following techniques. Two leaves of approximately the same size were removed from the transgenic plants and 50 μg / ml kanamycin with 0.1% ethanol or 0.1% ethanol and 10 μM methoprene or phenoxycarb (or, if the transgenic plant contained this marker, RTI ID = 0.0 > g / ml < / RTI > The leaves are incubated for about 24 hours under the standard growth conditions described in Example 14. After inducing the compound, the leaves were homogenized in 500 μl of 100 mM KPO ₄ , 1 mM DTT, pH 7.8 buffer at 0 ° C to prepare a leaf extract. The extract is centrifuged for 5 minutes in Eppendorf microfuge at 4 ° C and stored at 0 ° C until assayed. The luciferase activity in each extract is measured using an Analytical Luminescence Model 2010 photometer and Promega Luciferase Assay System according to the manufacturer's instructions. Extract protein concentrations were determined using the Pierce BCA protein assay (Smith et al., Anal. Biochem. 150: 76-85). The luciferase value is expressed as optical units per 10 seconds at room temperature per 100 μg of extracted protein. As shown in Table 4 below, the treatment of phenoxycav increased the luciferase activity by 6.2-fold and the treatment of methoprene increased the luciferase activity by 25-fold.

Receptor Chemical ligand Luciferase activity (optical unit) none none 638 35S / VP16-USP Phenoxycarb 3,991 35S / VP16-USP Methoprene 15,790

Example 16 Screening of Novel Ligands Binding to USP Using Gene-Implanted Plant or Plant Cells Expressing USP or RXR Derivatives and Including Receptor-Regulated Receptors

Novel ligands for USP or RXR effective under plant cell environment can be found by using screening methods based on the expression of receptor-regulated reporters as target expression cassettes in transgenic plant or plant cells expressing appropriate receptor polypeptides . In this method, a test for reporter gene expression is carried out after the chemicals tested for their ability to mediate USP or RXR activation of target polypeptide expression are contacted with transplanted plants or plant cells at different concentrations. For example, 1) a transgenic plant or plant cell containing a GAL4-regulated luciferase reporter as a target expression cassette and a receptor expression cassette for GAL4-EcR and USP-VP16 was exposed to the test material, and Hamamatsu ), And 2) GAL4-EcR-Cl and VP16-RXR, which contain a GAL4-regulated GUS reporter as a target expression cassette and a receptor expression cassette. Transplanted plant or plant cells are exposed to the test substance and exposed to a test substance such as 5-bromo-4-chloro-3-indolyl beta -D-glucuronide or o-nitrophenyl- beta -D-glucuronide Can be compared to unexplored plants for their ability to catalyze cleavage of the pigment source substrate, and 3) transgenic plants or plant cells containing DR RE-regulated luciferase reporter and expression cassettes for VP16-USP can be testedExposed to a substance, and compared with an unexposed plant using a light amplifying device such as a Hamamatsu optical detection device. Preferred control groups that may be useful in the screening method include, but are not limited to, tebufenozide and methoprene. In each of the above examples, the greater the detection of the expression level of the target polypeptide in the presence of the test substance compared to the expression level in the absence of the test substance, the greater the extent of the test substance ' s sensitivity to USP or RXR, depending on the receptor expression cassette used in the method. Ligand. In this way, a test substance that is not previously known as a ligand for USP in the plant cell environment can be identified by itself, or a test substance presumed to be a ligand for USP in plant cell environment can be identified as such.

Example 17: Isolation of low basal activity receptor polypeptide mutants

Mutants in the ligand binding domain of the ultraspiracle receptor (USP) are obtained in vitro by PCR mutagenesis as described in Leung et al., Technique 1: 11-15 (1989) . The PCR fragment of the mutated USP ligand binding domain is cloned into a yeast expression vector operatively linked to the transcriptional activation domain of VP16. The mutant construct is transformed into the yeast GAL4 reporter strain GGY1 :: 171. Yeast transformants are plated onto a medium containing indicator X-Gal. Mutants with reduced basal levels of USP receptor polypeptide activity on heterozygous dimers appeared as white to light blue colonies on X-Gal indicator plates, whereas transformants expressing non-mutated USP receptor polypeptides were found to be cancerous Blue colonies. White to light blue colonies are tested for basal and chemical ligand-induced levels of receptor polypeptide activity by growing yeast cells expressing these selected colonies in S medium containing glycerol, ethanol and galactose as a carbon source. The resulting culture is divided into two aliquots, one of which is treated with the larval hormone or its agonist and the other is used as a control in the absence of the chemical ligand. After exposure to the larval hormone or its agonist, both the treated fraction and the control fraction of the culture were subjected to Miller's process (Experiments in Molecular Genetics, p. 352-355, J.H. Miller, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1972). The nucleotide sequences encoding the mutant receptor polypeptides isolated and identified by the present technique are provided in another test because they show that the receptor polypeptides they encode exhibit a reduction in basal activity in plant cells, Of the target gene expression, or different agonists of the larval hormone.

Example 18 Identification of Functionally Improved Mutant Receptor Polypeptides in Plant Cells

A receptor expression cassette encoding the mutated USP receptor polypeptide of Example 15 is prepared according to Examples 2 and 4 above. These receptor expression cassettes are then transformed into plant cells according to the process of Example 9, in combination with the target expression cassette of Example 8. Transgenic plant cells are tested for activation of the 5'-regulated region of the target expression cassette by a mutant receptor polypeptide in the presence of the larval hormone or agonist thereof. Mutant USP receptor polypeptides that exhibit a low basal expression of the target polypeptide in the absence of chemical ligands in plant cells and high expression of the target polypeptide in the presence of the larval hormone or agonist thereof are useful for controlling gene expression in plants.

Example 19: Growth of transgenic plants

Transformed plants of Arabidopsis thaliana (Columbia) prepared in Example 14 were seeded in a 3-in- ² plastic pot in an artificial gas chamber set at 20 ° C for 16 hours and 8 hours for 4 to 5 weeks, And grown in coated soil. Plants contain DNA integrated into their genomic DNA in the form of a receptor according to the invention and a target expression cassette. The integrated DNA is transferred from one generation to the next through the modification process as a result of the life cycle of the transformed plant. Modification is the process by which male gametophytes and sporozoites or gametocyte tissues interact to form successful conjugates. Mature pollen particles are generated from the flower's medicine and deposited (moisture) on the surface of the head, hydrated and germinated to grow the pollen tube. The spermatids in the flowerpot are transmitted to the backpack (magnetic element) present in the ovary, and the actual event of fertilization (genital fusion) occurs to produce a conjugate. The seed-shaped conjugate realizes the next generation of the plant system. This next generation is called the "offspring" of the transformed plant. The offspring can be formed by self-fertilization, where male gametophytes and magnetic gametes occur in the same plant. This means that a single plant is a source of genomic DNA for the next generation. On the other hand, offspring can be produced by hybridization of two separate plants by contacting the male gametophytes from arbitrary plants with the magnetic sporophyte tissue of the separate plant to produce the next generation of plants. In this case, the genomic DNA of the offspring is derived from two separate plants. In addition, when the transformed plant is hybridized with non-transformed plants, the genomic DNA of the offspring consists of genetically-engineered genomic DNA from one plant and non-transgenic genomic DNA from isolated plants. Regardless of whether the offspring of the transgenic plant are produced by self-fertilization or cross-fertilization, some of the offspring will be given a different genetic sharing structure due to the presence of exogenous DNA incorporated into the genome. These different genetic sharing structures can be identified using the techniques of classical genetics and molecular biology.

To produce the next generation of plants containing the receptor and the target expression cassette according to the invention, the original transformed plants are matured and blossomed to produce seeds under controlled environmental conditions. The seeds obtained are dried in a drying chamber at 95 < 0 > F and low humidity for about 5 to 10 days. The filament is pulverized and then filtered through a 425 mu m mesh sieve to remove seeds from the dried flower to separate seeds from other plant materials. Seeds can then be used to grow the next generation of plants.

Although described for Arabidopsis, this method of producing the next generation of transformed plants is generally applicable to all pizza plants incorporating the receptors and target expression cassettes according to the present invention into their genomes.

All publications and patent applications mentioned in this application are indicative of the level of skill of those of ordinary skill in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent source was specifically and individually indicated to be incorporated by reference.

While the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

Sequence List

(1) General information:

(i) Applicant:

(A) Name: Shiva-kai

(B) Distance: Klebekstrasse 141

(C) City: Basel

(E) Country: Switzerland

(F) Postal code: 4002

(G) Phone number: +41 61 69 11 11

(H) Tel Fax: +41 61 696 79 76

(I) Telex: 962 991

(ii) Title of the invention: Lipid hormone or its agonist as a chemical ligand for regulating gene expression in plants by receptor mediated transactivation

(iii) SEQ ID NO: 11

(iv) Computer readable form:

(A) Media type: floppy disk

(B) Computer: IBM PC compatible

(C) Operating System: PC-DOS / MS-DOS

(D) Software: PatentIn Release # 1.0, Version # 1.30B

(2) Information on sequence 1:

(i) Sequence characteristics:

(A) Sequence length: 42 base pairs

(B) type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: Straight-line

(ii) Molecular type: other nucleic acid

(A) Description: / desc = "oligonucleotide SF43"

(iii) Hypothesis: None

(xi) Sequence description for sequence 1:

CGCGGATCCT AAACAATGAA GCGGCGCTGG TCGAACAACG GC 42

(2) Information on SEQ ID NO: 2:

(i) Sequence characteristics:

(A) Sequence length: 34 base pairs

(B) type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: Straight-line

(ii) Molecular type: other nucleic acid

(A) Description: / desc = "oligonucleotide SF23"

(iii) Hypothesis: None

(xi) Sequence description for sequence 2:

CGCGGGATCC ATGCGGCCGG AATGCGTCGT CCCG 34

(2) Information on SEQ ID NO: 3:

(i) Sequence characteristics:

(A) Sequence length: 26 base pairs

(B) type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: Straight-line

(ii) Molecular type: other nucleic acid

(A) Description: A positive sequence oligonucleotide used to construct /desc="pSKGAL2.3 "

(iii) Hypothesis: None

(xi) Sequence description for SEQ ID NO: 3:

CGGGGGATCC TAAGTAAGTA AGGTAC 26

(2) Information on SEQ ID NO: 4:

(i) Sequence characteristics:

(A) Sequence length: 20 base pairs

(B) type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: Straight-line

(ii) Molecular type: other nucleic acid

(A) Description: A complementary oligonucleotide used to construct /desc="pSKGAL2.3 "

(iii) Hypothesis: None

(xi) Sequence description for sequence 4:

CTTACTTACT TAGGATCCCC 20

(2) Information on SEQ ID NO: 5:

(i) Sequence characteristics:

(A) Sequence length: 30 base pairs

(B) type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: Straight-line

(ii) Molecular type: other nucleic acid

(A) Description: / desc = "oligonucleotide SF42"

(iii) Hypothesis: None

(xi) Sequence description for sequence 5:

CGCGGATCCA TGGACAACTG CGACCAGGAC 30

(2) Information on SEQ ID NO: 6:

(i) Sequence characteristics:

(A) Sequence length: 29 base pairs

(B) type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: Straight-line

(ii) Molecular type: other nucleic acid

(A) Description: / desc = "oligonucleotide SF37"

(iii) Hypothesis: None

(xi) Sequence description for sequence 6:

GCGGGATCCC CCACCGTACT CGTCAATTC 29

(2) Information on SEQ ID NO: 7:

(i) Sequence characteristics:

(A) Sequence length: 45 base pairs

(B) type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: Straight-line

(ii) Molecular type: other nucleic acid

(A) Description: / desc = "Oligonucleotide SA115"

(iii) Hypothesis: None

(xi) Sequence description for sequence 7:

GTCGAGCTCT CGGATCCTAA AACAATGGCC CCCCCGACCG ATGTC 45

(2) Information about SEQ ID NO: 8:

(i) Sequence characteristics:

(A) Sequence length: 35 base pairs

(B) type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: Straight-line

(ii) Molecular type: other nucleic acid

(A) Description: / desc = "oligonucleotide SF30"

(iii) Hypothesis: None

(xi) Sequence description for sequence 8:

CGCGGATCCA TGGGTCGCGA TGATCTCTCG CCTTC 35

(2) Information on SEQ ID NO: 9:

(i) Sequence characteristics:

(A) Sequence length: 16 amino acids

(B) Type of sequence: Amino acid

(C) Number of chains: 1 chain

(D) Topology: Straight-line

(ii) Type of molecule: Peptide

(iii) Hypothesis: None

(v) Short Type: Internal

(xi) Sequence description for sequence 9:

Val Pro Gly Pro Pro Ser Ser Arg Ser Val Ser Ile Ser Leu His Ala

1 5 10 15

(2) Information on SEQ ID NO: 10:

(i) Sequence characteristics:

(A) Sequence length: 30 base pairs

(B) type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: Straight-line

(ii) Molecular type: other nucleic acid

(A) Description: / desc = "Primer SF77"

(iii) Hypothesis: None

(xi) Sequence description for sequence 10:

GATCCGTAGG GGTCACGAAG TTCACTCGCA 30

(2) Information on SEQ ID NO: 11:

(i) Sequence characteristics:

(A) Sequence length: 30 base pairs

(B) type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: Straight-line

(ii) Molecular type: other nucleic acid

(A) Description: / desc = "Primer SF78"

(iii) Hypothesis: None

(xi) Sequence description for SEQ ID NO: 11:

GATCTGCGAG TGAACTTCGT GACCCCTACG 30

Claims (31)

a) a USP receptor expression cassette encoding a USP (Ultraspiracle) receptor polypeptide and b) a transgenic plant cell, food material or plant and its offspring comprising a target expression cassette encoding the target polypeptide. The plant cell, food material or plant according to claim 1, wherein the expression of the target expression cassette interferes with the plant modification. Plant cell or plant is a) a USP receptor expression cassette encoding a USP receptor polypeptide and b) transforming the plant cell or plant according to claim 1 with a target expression cassette encoding the target polypeptide. 4. The method of claim 3, comprising obtaining a plant cell or a progeny of a plant transformed with an expression cassette. The plant according to claim 1, which is corn. 2. The plant according to claim 1, a) expressing a receptor polypeptide in the plant and b) additionally comprising a second receptor expression cassette encoding a second receptor polypeptide different from the plant or USP receptor polypeptide according to claim 1, comprising contacting the plant with a juvenile hormone or an agonist thereof To regulate gene expression in plants. 8. The method of claim 7, wherein the expression of the target polypeptide is increased or activated in the presence of the nematicide or agonist thereof. 8. The method of claim 7, wherein the expression of the target polypeptide is reduced or suppressed in the presence of a nematicide or an agonist thereof. 8. The method of claim 7, wherein the USP receptor polypeptide comprises a heterologous transactivation domain. 11. The method of claim 10, wherein the heterologous transactivation domain is a transactivation domain from the VP16 protein of herpes simplex. 8. The method of claim 7, wherein the second receptor polypeptide is selected from the group consisting of EcR, DHR38, and RXR. 8. The method of claim 7, wherein the agonist is selected from the group consisting of phenoxycarb, diophenolol, quinoprene, methopren, hydrofren, diophenolol, methoprenic acid, triflumuron, hexamflumuron, teflubenzuron, flufenoxuron, ≪ / RTI > cyclodextrins, cyclosuurons, and leupenuron, diffluenzuron, and chlorflualuron. 8. The method of claim 7, wherein the target expression cassette comprises a 5 'regulatory region comprising from 1 to 11 copies of a response element. a) expressing a receptor polypeptide or polypeptides in the plant and b) regulating the plant modification, wherein the plant further comprises a second receptor expression cassette encoding a plant or a second receptor polypeptide according to claim 2, comprising contacting the plant with an insect hormone or an agonist thereof. 16. The method of claim 15, wherein the expression of the target polypeptide is increased or activated in the presence of a nematicide or agonist thereof. 16. The method of claim 15, wherein the expression of the target polypeptide is reduced or suppressed in the presence of the nematicide or agonist thereof. 16. The method of claim 15, wherein the USP or second receptor expression cassette comprises a anther-specific promoter operably linked to a coding sequence for a receptor polypeptide. 16. The method of claim 15, wherein the USP or second receptor expression cassette comprises a pistil-specific promoter operably linked to a coding sequence for a receptor polypeptide. 16. The method of claim 15, wherein the target polypeptide is ribonuclease vardenase. 16. The method of claim 15, wherein the target expression cassette encodes an anti-sense sequence that disrupts the modification. 16. The method of claim 15, wherein the target polypeptide restores an effective modification. 23. The method of claim 22, wherein the target polypeptide is a ribonuclease inhibitor Varustin. a) transforming the plant cell with a USP receptor expression cassette encoding a USP receptor polypeptide and a target expression cassette encoding the target polypeptide; b) expressing a USP receptor polypeptide in a plant cell; c) contacting the plant cell with the test substance; d) measuring the expression of the target polypeptide, and e) identifying a ligand of a USP receptor polypeptide that activates or inhibits the expression of the target expression cassette in a plant cell environment, comprising identifying a test substance that significantly activates or inhibits expression of the target polypeptide. 25. The method of claim 24, further comprising transforming the plant cell with a second receptor expression cassette encoding a second receptor polypeptide different from the USP receptor polypeptide and expressing a second receptor polypeptide. 25. The method of claim 24, wherein the expression of the target polypeptide is qualitatively determined. 25. The method of claim 24, wherein the expression of the target polypeptide is quantitatively determined. a) synthesizing a novel test substance according to a method known in the art; b) transforming the plant cell with a USP receptor expression cassette encoding a USP receptor polypeptide and a target expression cassette encoding the target polypeptide; c) culturing the offspring cells of the transformed plant cells; d) expressing a USP receptor polypeptide in offspring cells; e) contacting the offspring cells with the test material of step a); f) measuring the expression of the target polypeptide; g) repeating the process steps e) and f) using different test materials according to step a); h) selecting a test substance that significantly activates or inhibits the expression of the target polypeptide; and i) The method comprises repeating the method step a) for the selected material in step h), wherein the ligand of the USP receptor polypeptide is produced. 29. A ligand of a USP receptor obtainable by the method of claim 28. a) a USP receptor expression cassette encoding a USP receptor polypeptide and b) an agricultural method in which a transgenic material or plant containing a target expression cassette encoding a target polypeptide is used. Use of a larval hormone or agonist for modulating the expression of a target polypeptide in a plant according to claim 1.